Feature

The LEP proline-rich region (aa 81-105) showed a high score in ... the PESTfind analysis. Such PEST sequences, which are enriched for proline (P), glutamine (E), serine (S) and threonine (T), were found to be responsible for rapid proteolytic degradation

Feature

ANT has DNA binding abilities ... The consensus binding site has the sequence 5′-gCAC(A/G)N(A/T)TcCC(a/g)ANG(c/t)-3′ where the uppercase letters indicate the most highly conserved positions (present in >90% of the selected sites) and lowercase letters indicate somewhat less conserved positions (present in at least 65% of the selected sites). N indicates positions for which no particular base appeared to be preferred. The binding site is a 16 bp sequence containing 14 conserved positions

Feature

The NAC1 cDNA is 1272 bp in length and encodes a protein of 324 amino acids (Fig. ​(Fig.1A).1A). The N-terminal 172 residues contain the five conserved blocks of homology that characterize the NAC family. The divergent C terminus of 152 amino acids displays no homology to other known proteins

Feature

KAN consists of 6 exons, and encodes a protein with a 56 amino-acid domain (residues 220-275) that shares between 30% and 98% amino-acid identity with more than 50 predicted genes in the Arabidopsis genome, including several puta- tive transcription factors. This conserved domain has recently been named the GARP domain

Feature

The coding region of the cDNA encodes a protein of 476 amino acids that shares an overall identity of 28% with the budding yeast CAP (Figure 1) . The similarity was much higher (76%) with the cotton GhCAP. Therefore, the isolated cDNA was designated AtCAP1 (Arabidopsis thaliana CAP homolog

Feature

ANT ... The ability of several of these mutants to bind DNA in vitro was examined using gel mobility shift assays ... Five of the six proteins from mutants that gave a white colony-lift β-galactosidase phenotype showed significantly decreased DNA binding (Y318C, L319Q, L337P and D427G) or absolutely no DNA binding (R387G

Krizek BA - AINTEGUMENTA utilizes a mode of DNA recognition distinct from that used by proteins containing a single AP2 domain

Feature

Using this two-step screening procedure, 28 individual colonies were identified that expressed a full-length ANT protein but showed either reduced (four colonies) or no (24 colonies) β-galactosidase activity. Each clone had either one (19 clones) or two (nine clones) amino acid changes within the DNA-binding region of ANT (Table ​(Table1).1). Mutations in seven positions (V287, Y318, L319, D333, L337, F379 and S384) were represented multiple times within the collection

Krizek BA - AINTEGUMENTA utilizes a mode of DNA recognition distinct from that used by proteins containing a single AP2 domain

Feature

ANT ... One additional mutant was assayed in the BK1 system. The G382D mutation, corresponding to the molecular defect present in the strong ant-2 mutation, contains a replacement of a highly conserved glycine at the last position of the linker (2). Yeast expressing the G382D mutant gave a white phenotype in the colony-lift filter assay (Table ​(Table11

Krizek BA - AINTEGUMENTA utilizes a mode of DNA recognition distinct from that used by proteins containing a single AP2 domain

Feature

The deduced AtGRF proteins contain the highly conserved QLQ and WRC domains in their N-terminal region ... The QLQ domains of OsGRF1 and AtGRF proteins are characterized by the conserved Gln-Leu-Gln residues, the only exception being AtGRF9, which has Phe, a conservative substitution for Leu, in place of Leu (Figure 1b). Another feature of this domain is the absolute conservation of bulky aromatic/hydrophobic and acidic amino acid residues such as Phe, Trp, Tyr, Leu, Glu, or their equivalents in terms of chemical and radial properties. The Pro residue is also absolutely conserved ... The WRC domain of all the AtGRF proteins and of OsGRF1 contains two distinctive structural features, namely many basic amino acids (Arg and Lys) and the conserved spacing of three Cys and one His residues, the C3H motif (Figure 1c). The basic amino acids are highly conserved, not only in the AtGRFs but also in all the GRF homologs of other seed plants (Figure 1c; Table S1; http://www.theplantjournal.com), indicating that they are essential for the function of the WRC domain, probably as nuclear localization signal (Van der Knaap et al., 2000). The C3H motif is also absolutely conserved in the GRF homologs of Arabidopsis and of other seed plants

Kim JH, Choi D, Kende H - The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis

Feature

Overall, the HDZip I proteins share approximately 60% amino acid identity in the homeodomain, the most highly conserved part corresponding to the helix 3 region. In this comparison, ATHB16 shows a high degree of sequence similarity specifically to ATHB6 (Söderman et al., 1994); 93% amino acid identity over the homeodomain and 86% amino acid identity over the leucine zipper motif (Fig. 1

Feature

ATHB16 ... sequence contains a leucine zipper motif with 5 leucines and 1 isoleucine occurring in every seventh position, C-terminal to the homeodomain, in a position similar to those of previously known HDZip proteins

Feature

The UCU2 gene consists of eight exons, and the UCU2 protein shows significant similarity with the FKBP-type immunophillins (Galat, 2000), which are characterized by a conserved FKBP-like domain carrying the PPIase activity (Schiene-Fischer and Yu, 2001). When we compared the UCU2 amino acid sequence with those of already known FKBPs, we found a single PPIase domain that includes amino acids 58 to 156

Feature

All plant C2H2-type zinc finger proteins, including JAG, contain a conserved QALGGH sequence that has been shown in the SUP zinc finger to form part of an alpha-helix that contacts the major groove of DNA (Isernia et al., 2003

Feature

Database searches revealed 22 other putative Arabidopsis proteins with similarity to ROT4. These proteins were named ROT FOUR LIKE1�22 (RTFL1�22). Alignment revealed a 29-amino acid region that was conserved between the RTFL members (Figure 6a). The region was rich in basic amino acids, and had not been previously identified in any other proteins of known function. We named the novel region RTF domain

Feature

The Lissence- phaly type-1-like (LisH) motif, which is present in RON2 (LUG) (Fig. 5) is an a-helical motif with a putative function in the regulation of microtubuli dynamics (NCBI CDD SMART 00667.6, LISH)

Feature

The N-terminal domain (from residues 6 to 74 in RON2) is slightly longer than the LUFS domain predicted by Conner and Liu (2000) (Fig. 5), and is very conserved among plants. This domain is similar to the N-terminal region of the transcriptional activators FLO1 and FLO8 (Saccharomyces cerevisiae) and the members of the family of single-stranded DNA- binding proteins described in human, chicken, mouse, frog, zebrafish, and fruit fly (Castro et al., 2002

Feature

Sequence homology searches and phylogenetic analysis across the entire AP2/EREBP family showed that SHN1/WIN1 is part of a small, distinct group of three proteins, 199, 189, and 186 amino acid residues long (SHN1/WIN1, SHN2, and SHN3, respectively; Figure 6). They contain the highly conserved AP2 domain and share two other conserved motifs in their central portion (mm in Figure 6A) and C termini (cm in Figure 6A). The two complete motifs outside the AP2 domain are only present in the SHN clade proteins, whereas their next Arabidopsis homolog (At5g25190) contains only part of the mm domain and the cm domain

Feature

Sequence homology searches and phylogenetic analysis across the entire AP2/EREBP family showed that SHN1/WIN1 is part of a small, distinct group of three proteins, 199, 189, and 186 amino acid residues long (SHN1/WIN1, SHN2, and SHN3, respectively; Figure 6). They contain the highly conserved AP2 domain and share two other conserved motifs in their central portion (mm in Figure 6A) and C termini (cm in Figure 6A). The two complete motifs outside the AP2 domain are only present in the SHN clade proteins, whereas their next Arabidopsis homolog (At5g25190) contains only part of the mm domain and the cm domain

Feature

Sequence homology searches and phylogenetic analysis across the entire AP2/EREBP family showed that SHN1/WIN1 is part of a small, distinct group of three proteins, 199, 189, and 186 amino acid residues long (SHN1/WIN1, SHN2, and SHN3, respectively; Figure 6). They contain the highly conserved AP2 domain and share two other conserved motifs in their central portion (mm in Figure 6A) and C termini (cm in Figure 6A). The two complete motifs outside the AP2 domain are only present in the SHN clade proteins, whereas their next Arabidopsis homolog (At5g25190) contains only part of the mm domain and the cm domain

Feature

GIF proteins and SYT share an unusually high frequency of Gln (19% in SYT and 13–17% in GIFs) and Gly (15% in SYT and 10–15% inGIFs). TheGIF proteins, however, lack the repetitive occurrence of Pro and Tyr residues that are abundant in the QPGY domain of SYT (8). Therefore, we call the Gln/Gly-rich region of GIFs the QG domain

Feature

level of relatedness between SYT and the GIF proteins (Fig. 8). GIF proteins and SYT share an unusually high frequency of Gln (19% in SYT and 13–17% in GIFs) and Gly (15% in SYT and 10–15% inGIFs). TheGIF proteins, however, lack the repetitive occurrence of Pro and Tyr residues that are abundant in the QPGY domain of SYT (8). Therefore, we call the Gln/Gly-rich region of GIFs the QG domain

Feature

The second conserved domain is a series of four ankyrin repeats (Figure 1D), which in NPR1 have been shown to interact with members of the TGA family of transcription factors (Zhang et al., 1999; Despr�s et al., 2000; Zhou et al., 2000

Feature

The CCHC zinc finger motif (CX2CX4HX4C ... SMP1

Clay NK, Nelson T - The recessive epigenetic swellmap mutation affects the expression of two step II splicing factors required for the transcription of the cell proliferation gene STRUWWELPETER and for the timing of cell cycle arrest in the Arabidopsis leaf

Feature

The CCHC zinc finger motif (CX2CX4HX4C ... SMP2

Clay NK, Nelson T - The recessive epigenetic swellmap mutation affects the expression of two step II splicing factors required for the transcription of the cell proliferation gene STRUWWELPETER and for the timing of cell cycle arrest in the Arabidopsis leaf

Feature

(At1g65660) encodes a CCHC zinc finger protein

Clay NK, Nelson T - The recessive epigenetic swellmap mutation affects the expression of two step II splicing factors required for the transcription of the cell proliferation gene STRUWWELPETER and for the timing of cell cycle arrest in the Arabidopsis leaf

Feature

ATAF2 (At5g08790) is a 1032 base pairs (bp) gene containing two introns, which encodes a 283 amino acids long protein. Its genomic structure is typical for NAC-domain proteins with the first two exons and the 5′-end of the third exon encoding the NAC-domain, while the rest of the gene encodes the non-conserved C-terminal region

Feature

The location of the GTE6 protein in the cell was examined using Arabidopsis plants stably transformed with a 35S::GTE6-GFP construct, which expresses a GTE6-green fluorescent protein (GFP) fusion protein. The GTE6-GFP fusion protein is localized to the nucleus (Fig. 6A), which is consistent with its predicted role as a transcription factor

Feature

Following the complete sequencing of the Arabidopsis genome, GTE6 was annotated as a BET (bromodomain-Extraterminal) gene, because it contains a bromodomain and an Extraterminal (ET) domain, and was classified as a member of the GTE family of transcription factors

Feature

Recently, a family of plant-specific zinc finger homeodomain (ZF-HD) proteins was identified from in vitro analysis of plant DNA-binding proteins (Windhovel et al., 2001). During the process of studying the Arabidopsis members of this ZF-HD family, we identified a gene (At1g74660) that contains the putative zinc finger (ZF) but not the homeodomain

Feature

The MIF1 gene (At1g74660) corresponds to a 551-bp mRNA sequence (GenBank accession number AY085327) and encodes a putative protein of 101 amino acid residues (Figure 1a). It is highly similar to the N-terminal region of ZF-HD proteins (Figure 1b). The putative ZF domain contains at least eight cysteine and/or histidine residues (CX3HX11CX12–26CX2CXCHX3H) (Figure 1c). The spacing pattern of cysteine and histidine residues in MIF1 and ZF-HD proteins is different from that of other known zinc finger domains, such as C2H2 and RING fingers

Feature

According to the annotations in the databases, BDG belongs to the α/β-hydrolase fold superfamily of proteins and contains the predicted hydrolase/acyltransferase domain (KOG1454). Members of this superfamily (hereafter referred to as α/β-hydrolases) show little amino acid sequence similarity, with the exception of the conserved catalytic triad composed of a base (His), a nucleophile (Ser), and an acid (Asp/Glu)

Feature

CLE44 ... differ from the consensus at positions 3 and 13, with His in place of Arg at position 3 and Asn in place of His at position 13 ... Despite their divergence from two of the best-conserved amino acids in the CLE domain, CLE41, 42, and 44 all have an uninterrupted core of seven conserved amino acids (positions 5–11) and are less divergent from the main consensus than CLE7

Feature

CLE41 ... differ from the consensus at positions 3 and 13, with His in place of Arg at position 3 and Asn in place of His at position 13 ... Despite their divergence from two of the best-conserved amino acids in the CLE domain, CLE41, 42, and 44 all have an uninterrupted core of seven conserved amino acids (positions 5–11) and are less divergent from the main consensus than CLE7

Feature

To determine whether the amino terminal half of ANT was sufficient for transcriptional activation in yeast, a fusion of the GAL4 DNA binding domain to the first 274 amino acids of ANT (GBD–ANT1–274) was made (Fig. 1b). High β-galactosidase activity was present in yeast cells expressing GBD–ANT1–274 (Fig. 1b), demonstrating that the amino terminal half of ANT is both necessary and sufficient for the transcription activation function of ANT in yeast

Feature

To map the region of ANT required for this transcriptional activation function, amino terminal truncated forms of ANT were fused to the GAL4 DNA binding domain (Fig. 1b). In GBD–ANT133–555, the amino terminal 132 amino acids have been deleted. In GBD–ANTΔ41–53, the Ser-rich region has been removed. In GBD–ANTΔ214–231, the Gln, Asn, His-rich region has been removed. GBD–ANTΔ41–53,Δ214–231 corresponds to a protein lacking the Ser-rich and Gln, Asn, His-rich regions, while GBD–ANT275–555 lacks the first 274 amino acids of ANT. GBD–ANT133–555, GBD–ANTΔ41–53, GBD–ANTΔ214–231, and GBD–ANTΔ41–53, Δ214–231 all produced high levels of β-galactosidase activity, while GBD–ANT275–555 showed almost no β-galactosidase activity (Fig. 1b). These results suggest that the amino terminal half of ANT is required for transcriptional activation in yeast

Feature

Amino acids 252–255 of ANT, which correspond to the sequence KKKR, have been suggested previously to encode a nuclear localization signal (Klucher et al. 1996). To investigate the importance of this sequence for the subcellular localization of ANT, Lys 253 and Lys 255 were mutated to Thr. GFP–ANTK253T,K255T was present in both the cytoplasm and nucleus of bombarded leek epidermal cells (Fig. 6c). This suggests that the basic stretch of amino acids at positions 252–255 is a nuclear localization signal

Feature

To further define the transcriptional activation domain of ANT, smaller regions of the amino terminal half of ANT were fused to the GAL4 DBD. GBD–ANT1–133 corresponding to the amino terminal 133 residues was not able to activate lacZ expression (Fig. 1b). GBD–ANT133–274 and GBD–ANT134–213 produced high levels of β-galactosidase activity (Fig. 1b). This suggests that an 80 amino acid region between residues 134 and 213 is critical for transcriptional activation. Yeast containing GBD–ANTΔ134–213, which has a deletion of this 80 amino acid region, produced very low levels of β-galactosidase activity

Feature

Deletion analysis of the FIL promoter identified two cis-acting regulatory elements required for proper FIL expression (Watanabe and Okada, 2003). A region proximal to the FIL coding sequence (?1,742 to ?1,547) is required for expression in both adaxial and abaxial domains, while a 12-bp (−1,748 to −1,737) sequence is required for the abaxial-specific expression of the FIL gene

Feature

The RE gene ... would produce a protein of 348 amino acids ... The attempts to identify the At2g37860.1 transcript were not successful, suggesting that it is either artefactual, unstable, produced in developmental stages other than those studied here, or strictly restricted to a reduced number of cells during development

Feature

This intronless gene, henceforth named BOLITA (BOL), was predicted to encode a 306 aa protein that belongs to the ERF family, as it contains a single AP2/ERF domain. The closest homolog of BOL in the Arabidopsis genome is DRN/ESR1, which led to it being referred to as DRNlike

Feature

HUB1 contains a RING finger domain (PF00097) at position 826 to 864 and is classified as an HCa- RING-type protein (Stone et al., 2005). RING finger domains are specialized types of Zn finger domains of 40 to 60 residues that bind two atoms of zinc and are known to mediate protein–protein interactions

Feature

An acidic/Ser-rich region (45% Ser, 34% aspartic or glutamaic acid in yeast NSR1, and 24% and 32% in PARL1) resides in the amino-terminal part of the protein (amino acids 60–263), followed by two RNA recognition motifs (RRMs; amino acids 298–370 and 402– 477, respectively) and a carboxyl-terminal Gly- and Arg-rich (GAR) domain (62% Arg or Gly in NSR1 and 69% in PARL1, amino acids 481–549; Fig. 2B

Feature

STRUBBELIG-RECEPTOR FAMILY (SRF) ... members ... encode putative LRR-RLKs with an extra-cellular domain (ECD), a transmembrane domain (TM), an intracellular juxtamembrane domain (JM), an intracellular catalytic or kinase domain (CD), and in some cases, an extended C-terminus

Feature

STRUBBELIG-RECEPTOR FAMILY (SRF) ... members ... encode putative LRR-RLKs with an extra-cellular domain (ECD), a transmembrane domain (TM), an intracellular juxtamembrane domain (JM), an intracellular catalytic or kinase domain (CD), and in some cases, an extended C-terminus

Feature

STRUBBELIG-RECEPTOR FAMILY (SRF) ... members ... encode putative LRR-RLKs with an extra-cellular domain (ECD), a transmembrane domain (TM), an intracellular juxtamembrane domain (JM), an intracellular catalytic or kinase domain (CD), and in some cases, an extended C-terminus

Feature

STRUBBELIG-RECEPTOR FAMILY (SRF) ... members ... encode putative LRR-RLKs with an extra-cellular domain (ECD), a transmembrane domain (TM), an intracellular juxtamembrane domain (JM), an intracellular catalytic or kinase domain (CD), and in some cases, an extended C-terminus

Feature

STRUBBELIG-RECEPTOR FAMILY (SRF ... members ... encode putative LRR-RLKs with an extra-cellular domain (ECD), a transmembrane domain (TM), an intracellular juxtamembrane domain (JM), an intracellular catalytic or kinase domain (CD), and in some cases, an extended C-terminus

Feature

STRUBBELIG-RECEPTOR FAMILY (SRF) ... members ... encode putative LRR-RLKs with an extra-cellular domain (ECD), a transmembrane domain (TM), an intracellular juxtamembrane domain (JM), an intracellular catalytic or kinase domain (CD), and in some cases, an extended C-terminus

Feature

STRUBBELIG-RECEPTOR FAMILY (SRF) ... members ... encode putative LRR-RLKs with an extra-cellular domain (ECD), a transmembrane domain (TM), an intracellular juxtamembrane domain (JM), an intracellular catalytic or kinase domain (CD), and in some cases, an extended C-terminus

Feature

The four F-box proteins AT1G47056, AT3G50080, AT4G07400, and AT5G67250 share significant sequence similarity (56 to 69% identity and 63 to 80% similarity), and together they form a distinct F-box protein family within the C subfamily of the Arabidopsis F-box protein superfamily (Figures 1A and 1C) (Gagne et al., 2002). We named this protein family VFB and designated its individual members ... Common features of these F-box proteins are the presence of an N-terminal F-box domain and a series of LRRs, which are predicted to interact with the respective degradation substrates

Feature

The four F-box proteins AT1G47056, AT3G50080, AT4G07400, and AT5G67250 share significant sequence similarity (56 to 69% identity and 63 to 80% similarity), and together they form a distinct F-box protein family within the C subfamily of the Arabidopsis F-box protein superfamily (Figures 1A and 1C) (Gagne et al., 2002). We named this protein family VFB and designated its individual members ... Common features of these F-box proteins are the presence of an N-terminal F-box domain and a series of LRRs, which are predicted to interact with the respective degradation substrates

Feature

The four F-box proteins AT1G47056, AT3G50080, AT4G07400, and AT5G67250 share significant sequence similarity (56 to 69% identity and 63 to 80% similarity), and together they form a distinct F-box protein family within the C subfamily of the Arabidopsis F-box protein superfamily (Figures 1A and 1C) (Gagne et al., 2002). We named this protein family VFB and designated its individual members ... Common features of these F-box proteins are the presence of an N-terminal F-box domain and a series of LRRs, which are predicted to interact with the respective degradation substrates

Feature

The four F-box proteins AT1G47056, AT3G50080, AT4G07400, and AT5G67250 share significant sequence similarity (56 to 69% identity and 63 to 80% similarity), and together they form a distinct F-box protein family within the C subfamily of the Arabidopsis F-box protein superfamily (Figures 1A and 1C) (Gagne et al., 2002). We named this protein family VFB and designated its individual members ... Common features of these F-box proteins are the presence of an N-terminal F-box domain and a series of LRRs, which are predicted to interact with the respective degradation substrates

Feature

Database searches indicated that BEN1 belongs to a small gene family that also includes the well-characterized dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (BANYULS or BAN) (Figure 4a,b). Both play key roles in flavonoid biosynthesis pathways. BEN1 shares 42% sequence identity with DFR (At5g42800), 37% with both BAN (At1g61720) and At4g35420, and 34% with At4g27250. The closest homolog of BEN1 is DFR, as shown by phylogenetic analysis and amino acid alignment results (Figure 4a,b). Further analysis showed that proteins of this group contain a putative NADPH-binding domain, as well as a domain determining substrate specificity

Feature

Other motifs that are present in the BIN4 protein include a putative nuclear localization signal (KRGRPSKEKQPPAKKAR) located in the C-terminal part of the protein, suggesting that BIN4 might function within the nucleus

Feature

A PHI-BLAST sequence search revealed that BIN4 is a single-copy gene in Arabidopsis and appears to be plant-specific. The predicted BIN4 protein does not show strong similarity to any protein in public databases, except that its C terminus contains short sequences that are similar to the DNA binding domain of a High-Mobility Group protein (see Supplemental Figure 4A online). Importantly, the corresponding sequences in BIN4 possess an RGR motif, also called an AT hook, that is found in most High-Mobility Group proteins and shows strong binding to AT-rich DNA sequences (see Supplemental Figure 4D online

Feature

containing a UBP domain, with two short but well-conserved motifs, known as cysteine (Cys) and histidine (His) boxes, which include a triad of catalytic residues (Cys in the Cys box, His and Asp/Asn in the His box) ... UBP15

Liu Y, Wang F, Zhang H, He H, Ma L, Deng XW - Functional characterization of the Arabidopsis ubiquitin-specific protease gene family reveals specific role and redundancy of individual members in development

Feature

We then dissected the CDKA;1 promoter and observed GUS expression in tissues ... Despite the disappearance of GUS expression in roots, we could observe GUS signals in young leaves of the 7506R and 7507R lines (Fig. 4B), indicating the presence of root-specific regulatory elements that promote CDKA;1 expression

Feature

We then dissected the CDKA;1 promoter and observed GUS expression in tissues ... we created another reporter construct, 7509F/7R, which carries the region between − 890 bp and − 601 bp (Fig. 2). Although we included the region from − 890 bp to − 791 bp that promotes CDKA;1 expression as mentioned above, the GUS signal was very weak as compared to that in 7507R. However, we could again identify epidermis-specific expression (Fig. 5D), suggesting that this region contains enough information to promote epidermal expression in leaves

Feature

We then dissected the CDKA;1 promoter and observed GUS expression in tissues ... the 7-day-old seedlings, we found a significant decrease in the level of GUS expression between the 7502R and 7503R constructs (Fig. 4A). A similar reduction was also noted in embryos (Supplemental Fig. 3), indicating the presence of another cis-regulatory element between − 200 and − 101 bp ... 7503R exhibited lower GUS expression in the SAM and no expression in the inner layers of leaves. The results showed that the epidermis of leaves and trichomes were prominently GUS-stained (Fig. 5C). This indicates that the region between − 200 bp and − 101 bp is associated with CDKA;1 expression in the SAM and in leaves except for the epidermis

Feature

We then dissected the CDKA;1 promoter and observed GUS expression in tissues ... These results suggest that the region from − 890 bp to − 791 bp contains cis-regulatory element(s) that promote CDKA;1 expression independently of tissues

Feature

The SPI gene encodes a protein with a predicted length of 3600 amino acids and a molecular weight of approximately 400�kDa. An investigation of the primary structure of the predicted protein sequence yielded three common motifs. PrositeScan analysis revealed a pleckstrin homology (PH) domain (amino acids 2829�2927) adjacent to a BEACH domain (amino acids 2952�3244), and four WD40 repeats (amino acids 3326�3417

Feature

The SPI gene encodes a protein with a predicted length of 3600 amino acids and a molecular weight of approximately 400�kDa. An investigation of the primary structure of the predicted protein sequence yielded three common motifs. PrositeScan analysis revealed a pleckstrin homology (PH) domain (amino acids 2829�2927) adjacent to a BEACH domain (amino acids 2952�3244), and four WD40 repeats (amino acids 3326�3417

Feature

The SPI gene encodes a protein with a predicted length of 3600 amino acids and a molecular weight of approximately 400�kDa. An investigation of the primary structure of the predicted protein sequence yielded three common motifs. PrositeScan analysis revealed a pleckstrin homology (PH) domain (amino acids 2829�2927) adjacent to a BEACH domain (amino acids 2952�3244), and four WD40 repeats (amino acids 3326�3417

Feature

OLI2 encodes a protein related to Saccharomyces cerevi- siae, Nop2. Both Nop2 and its putative human ortholog, p120, localize in the nucleolus; upon growth stimulation, these proteins show high accumulation (de Beus et al., 1994). Nop2 is an essential protein that participates in the processing of 27S pre-rRNA into 5.8S and 25S rRNA, and probably in biogenesis of the 60S ribosome subunit (de Beus et al., 1994; Hong et al., 1997, 2001). Although the molecular function of Nop2 has not yet been conclusively demonstrated, it has been suggested to be an RNA m(5)C methyltransferase, based on the similarity of its C-terminal half to a m(5)C methyltransferase, Fmu, from Escherichia coli (de Beus et al., 1994; Hong et al., 1997, 2001; King et al., 1999; Tscherne et al., 1999). The known RNA and DNA methyltransferases have up to ten conserved motifs (Foster et al., 2003). In OLI2, Nop2, p120 and Fmu, motifs I, IV, VI and VIII are particularly well conserved (Figure S7). Motif I is part of the S-adenosyl- methionine binding pocket. Motifs IV and VI are within the catalytic site, and two invariant Cys residues in each motif are also conserved in OLI2

Feature

A detailed motif analysis of the RPT2 protein sequence confirmed that well-known RPT motifs are conserved in both AtRPT2 paralogs, including the ATP/GTP-binding site P-loop (motif A), the AAA-protein family signature (motif B) and a putative nuclear localization signal (NLS)

Feature

share a high amino acid sequence similarity that differs by only three amino acid residues (Figure 1a). A detailed motif analysis of the RPT2 protein sequence confirmed that well-known RPT motifs are conserved in both AtRPT2 paralogs, including the ATP/GTP-binding site P-loop (motif A), the AAA-protein family signature (motif B) and a putative nuclear localization signal (NLS)

Feature

Because bop1-1 plants exhibited dominant-negative phenotypes similar to those of bop1-4 bop2-11 plants (Ha et al., 2004), we assessed whether mutant bop1-1 protein could inhibit wild-type BOP transactivation activity. bop1-1 protein fused to the Gal4 BD (BD-b1-1) showed no transactivation activity (Figure 1B), indicating that the addition of four amino acids to the C terminus of the BOP1 protein completely abolished its transcriptional activation capacity. In the presence of bop1-1 protein, the transactivation capacity of wild-type BOP1 and BOP2 protein was strongly reduced (Figure 1B). Thus, bop1-1 protein either interferes with the transactivation function of the wild-type BOP proteins or itself functions as a transcriptional inhibitor.

Feature

PSI-BLAST searches of plant databases using the protein sequences of mammalian VKORC1 detected homologs (22–24% identity) that are fused to a thioredoxin-like domain (henceforth, VKORC1-Trx-like) in angiosperms (dicots and monocots), gymnosperms, mosses and green algae (Figure S1). These plant VKORC1 homologs display the two pairs of cysteine residues that are strictly conserved in their mammalian and cyanobacterial counterparts (Figure S1). The predicted topology of the Arabidopsis protein showed that the first cysteine pair (C109/C116) is located in a soluble region just before the first transmembrane helix, and that the second one (C195/C198) – a CXXC redox center thought to correspond to the active site in mammalian VKORC1 (Tie and Stafford, 2008) – is located within the third transmembrane helix (Figures S1 and S2). A fourth transmembrane helix is predicted to join the VKORC1 domain to the soluble Trx-like moiety, which also contains a CXXC redox center (Figures S1 and S2). In addition, the plant VKORC1 homologs display highly diverged N-terminal pre-sequences that have the hallmark of chloroplast targeting peptides (Figure S1 ... At4g35760

Feature

As shown in Figure 2a, SIG6 has a conserved C-terminal region (CR) containing subregions 1.2–4.2 for basic sigma factor functions, and a long N-terminal unconserved region (unconserved region; UCR) (Fujiwara et al., 2000; Schweer et al., 2009). Constructs containing a series of deletions of the SIG6 domains were generated and tested for their interactions with DG1. The unconserved region domain of SIG6 was found to be sufficient for its interaction with DG1 (Figure 2b).

Feature

Next, we examined the interaction between the C-terminal region of DG1 and the unconserved region of SIG6 using a pull-down assay. To perform this assay, we expressed the N-terminal region of SIG6 fused with a glutathione-S-transferase (GST) tag and the C-terminal region of DG1 fused with a His tag in Escherichia coli BL21 (DE3). After incubating the GST-N-SIG6 proteins with the DNase-treated total protein extracts from wild-type Arabidopsis leaves, the proteins eluted from the GST-binding resin were examined by SDS-PAGE and western-blot analysis using an antibody specific for DG1. DG1 was detected when GST-N-SIG6 fusion proteins were used in the assay, but no protein was detected when only the total protein extract and the resin were used (Figure 2c). Reciprocally, SIG6 was detected when the His-C-DG1 fusion protein was used (Figure 2c). These results indicated a direct interaction between the C-terminal region of DG1 and the unconserved region of SIG6

Feature

Next, we examined the interaction between the C-terminal region of DG1 and the unconserved region of SIG6 using a pull-down assay. To perform this assay, we expressed the N-terminal region of SIG6 fused with a glutathione-S-transferase (GST) tag and the C-terminal region of DG1 fused with a His tag in Escherichia coli BL21 (DE3). After incubating the GST-N-SIG6 proteins with the DNase-treated total protein extracts from wild-type Arabidopsis leaves, the proteins eluted from the GST-binding resin were examined by SDS-PAGE and western-blot analysis using an antibody specific for DG1. DG1 was detected when GST-N-SIG6 fusion proteins were used in the assay, but no protein was detected when only the total protein extract and the resin were used (Figure 2c). Reciprocally, SIG6 was detected when the His-C-DG1 fusion protein was used (Figure 2c). These results indicated a direct interaction between the C-terminal region of DG1 and the unconserved region of SIG6

Feature

To investigate which region of DG1 is required for the interaction with SIG6, several DG1 truncations were generated and tested for their ability to interact with SIG6 in the yeast two-hybrid system. Our results showed that the C-terminal region of DG1 is responsible for the interaction with SIG6 (Figure 2b). However, the interaction was abolished when the CTR of DG1 was further shortened by 51 amino acids

Feature

Sequence analysis identified a conserved 5′-terminal oligopyrimidine tract (5′ TOP) motif in the 5′ UTR and two AUUUA motifs in the 3′ UTR of plant AtTCTP (Fig. S7 E and F). In animals, 5′ TOP- and CG-rich regions in the 5′ UTR or AUUUA motifs in the 3′ UTR have been reported as important for the control of TCTP translation (4

Feature

AtTCTP, 38% of its amino acids are identical to its Drosophila counterpart (Fig. S10). Many of the essential amino acids and domains known to be required for animal TCTP functions are conserved in AtTCTP. The rat TCTP C-terminal domain, shown to homodimerize in the yeast two-hybrid system (23), is conserved in AtTCTP (Leu122 to Cys168). Bimolecular fluorescence complementation (BiFC) experiments (Fig. S11A) demonstrated that AtTCTP or dTCTP were able to homodimerize in vivo. Furthermore, AtTCTP was able to interact with dTCTP, suggesting that despite the overall relatively divergent protein sequences, the homodimerization domains of AtTCTP and dTCTP are structurally and functionally conserved. In Drosophila, a substitution of Glu12 to Val renders dTCTP nonfunctional (8). We found that AtTCTP harboring such a mutation (AtTCTPE12V) was unable to complement eye and wing size reduction phenotypes associated with dTCTP loss of function in flies (Fig. 5 B–H), demonstrating that the Glu12 is necessary for the correct function of TCTP in both plants and animals

Feature

Except for EXO70B2pro::GUS transgenic plants that showed no GUS expression in the samples examined, for the other 22 EXO70 genes, distinct cellular expression levels were observed. There is little doubt that EXO70B2 is an expressed gene, as indicated by the microarray data (Supplemental Fig. S8; Chong et al., 2010), 31 ESTs found in The Arabidopsis Information Resource database, and our RT-PCR analysis. Since the distance between the start codon of EXO70B2 and that of the oppositely orientated upstream gene At1g070010 is only 643 bp, it is likely that regulatory elements are located beyond the 1,236-bp 5′ upstream sequence used

Feature

the FPGS1 and FPGS3 genes lie within regions that have undergone segmental chromosome duplication (Figures 2b and S2). Phylogenetic analyses of FPGS sequences from various angiosperms revealed the presence of two clades (Figure S3). The Arabidopsis FPGS1 and FPGS3 isoforms belong to clade I and are closely related, indicating that they evolved relatively recently by gene duplication

Feature

the FPGS1 and FPGS3 genes lie within regions that have undergone segmental chromosome duplication (Figures 2b and S2). Phylogenetic analyses of FPGS sequences from various angiosperms revealed the presence of two clades (Figure S3). The Arabidopsis FPGS1 and FPGS3 isoforms belong to clade I and are closely related, indicating that they evolved relatively recently by gene duplication

Feature

Representative MIXTA-like genes were isolated from Medicago truncatula (Mt), the asterid Antirrhinum majus (Am), and the monocot Dendrobium crumenatum (Dc) also known as pigeon orchid (see Figure S2a online). The relationships between the polypeptide sequences encoded by these MIXTA-like genes are shown in Figures 7 and S4 online. In Medicago, which contains trichomes with long unicellular spikes and smaller multi-cellular glandular trichomes (Damerval, 1983; Pang et al., 2009), three MIXTA-like (MtMYBML) genes were found. MtMYBML1 and 3 were more closely related to NOK ... The chosen MIXTA-like MYBs were expressed in nok trichomes and rescued the extra-branched nok phenotype (Figures 8a,c,e and S5). In addition, the three genes promoted trichome outgrowth and branch formation of gl3-sst nok trichomes (Figures 8b,d,f and S5). The best rescue of the double mutant was induced by the monocot DcMYBML1 (compare Figures 8f with 8b,d). The rescue was less dramatic with either MtMYBML3 or AmMYBML3, but enhanced branch formation and expansion were evident (compare Figure 8b,d with 3a), and some trichomes on these plants resembled those normally found on gl3-sst plants (compare Figure 8 b,d with 1g–i). t-test analyses of trichome size measurements from the non-transformed double mutant and from either of the AmMYBML3 or MtMYBML3 transformants showed that there were significant differences. Trichomes on both AmMYBML3 and MtMYBL3 transformants were 1.75-times larger than those on gl3-sst nok

Feature

In Arabidopsis, miR171 targets the transcripts of three GRAS gene family members (At2g45160, At3g60630 and At4g00150; Llave et al., 2002; Reinhart et al., 2002), which we named LOST MERISTEMS (LOM) genes based on the observed mutant phenotypes (see below). The LOM genes are related to the petunia HAIRY MERISTEM (PhHAM) gene (Tian et al., 2004), which has been shown to play an important role in maintaining the SAM in an undifferentiated state (Stuurman et al., 2002). However, PhHAM and the Arabidopsis LOM proteins showed only 36–38% sequence identity, and phylogenetic analysis demonstrated that they belong to different sub-clades

Feature

In Arabidopsis, miR171 targets the transcripts of three GRAS gene family members (At2g45160, At3g60630 and At4g00150; Llave et al., 2002; Reinhart et al., 2002), which we named LOST MERISTEMS (LOM) genes based on the observed mutant phenotypes (see below). The LOM genes are related to the petunia HAIRY MERISTEM (PhHAM) gene (Tian et al., 2004), which has been shown to play an important role in maintaining the SAM in an undifferentiated state (Stuurman et al., 2002). However, PhHAM and the Arabidopsis LOM proteins showed only 36–38% sequence identity, and phylogenetic analysis demonstrated that they belong to different sub-clades

Feature

In Arabidopsis, miR171 targets the transcripts of three GRAS gene family members (At2g45160, At3g60630 and At4g00150; Llave et al., 2002; Reinhart et al., 2002), which we named LOST MERISTEMS (LOM) genes based on the observed mutant phenotypes (see below). The LOM genes are related to the petunia HAIRY MERISTEM (PhHAM) gene (Tian et al., 2004), which has been shown to play an important role in maintaining the SAM in an undifferentiated state (Stuurman et al., 2002). However, PhHAM and the Arabidopsis LOM proteins showed only 36–38% sequence identity, and phylogenetic analysis demonstrated that they belong to different sub-clades

Feature

The proteins predicted to be encoded by the two Arabidopsis eIF6 genes share 86% sequence similarity at the amino acid level and are 72% identical (Supplemental Fig. S6, A and B). The protein sequence of eIF6A also appears to be highly conserved within the plant kingdom

Feature

Diploid S. cerevisiae strains of the genetic Σ1278b background are dimorph and develop from single spherical S. cerevisiae cells to filament-like pseudohyphal cells under nitrogen starvation conditions (Gimeno et al., 1992). A homozygous deletion of CPC2 results in the loss of pseudohyphae development under nitrogen starvation conditions and the formation of a smooth-border round colony (Fig. 3, A and B; Valerius et al., 2007). We first expressed the full-length S. cerevisiae CPC2 gene in the cpc2 mutant using the S. cerevisiae expression vector p424MET25 (Mumberg et al., 1994) and observed the restoration of pseudohyphae growth (Fig. 3C). With this validated system, we found that when any of the three Arabidopsis RACK1 genes were expressed in the S. cerevisiae cpc2 diploid mutant background, the transformant regained the ability to produce the filament-like structures (pseudohyphae; Fig. 3, D–F). These results demonstrated that the Arabidopsis RACK1 genes are functionally equivalent to the S. cerevisiae CPC2/RACK1. In an earlier study, Gerbasi et al. (2004) demonstrated that the mammalian RACK1 is also a functional ortholog of the S. cerevisiae CPC2 gene. In agreement with these genetic data, both the amino acid sequence (Chen et al., 2006) and crystal structure (Ullah et al., 2008) of RACK1 are also highly conserved in different eukaryotic organisms

Feature

The proteins predicted to be encoded by the two Arabidopsis eIF6 genes share 86% sequence similarity at the amino acid level and are 72% identical (Supplemental Fig. S6, A and B). The protein sequence of eIF6A also appears to be highly conserved within the plant kingdom

Feature

Diploid S. cerevisiae strains of the genetic Σ1278b background are dimorph and develop from single spherical S. cerevisiae cells to filament-like pseudohyphal cells under nitrogen starvation conditions (Gimeno et al., 1992). A homozygous deletion of CPC2 results in the loss of pseudohyphae development under nitrogen starvation conditions and the formation of a smooth-border round colony (Fig. 3, A and B; Valerius et al., 2007). We first expressed the full-length S. cerevisiae CPC2 gene in the cpc2 mutant using the S. cerevisiae expression vector p424MET25 (Mumberg et al., 1994) and observed the restoration of pseudohyphae growth (Fig. 3C). With this validated system, we found that when any of the three Arabidopsis RACK1 genes were expressed in the S. cerevisiae cpc2 diploid mutant background, the transformant regained the ability to produce the filament-like structures (pseudohyphae; Fig. 3, D–F). These results demonstrated that the Arabidopsis RACK1 genes are functionally equivalent to the S. cerevisiae CPC2/RACK1. In an earlier study, Gerbasi et al. (2004) demonstrated that the mammalian RACK1 is also a functional ortholog of the S. cerevisiae CPC2 gene. In agreement with these genetic data, both the amino acid sequence (Chen et al., 2006) and crystal structure (Ullah et al., 2008) of RACK1 are also highly conserved in different eukaryotic organisms

Feature

Diploid S. cerevisiae strains of the genetic Σ1278b background are dimorph and develop from single spherical S. cerevisiae cells to filament-like pseudohyphal cells under nitrogen starvation conditions (Gimeno et al., 1992). A homozygous deletion of CPC2 results in the loss of pseudohyphae development under nitrogen starvation conditions and the formation of a smooth-border round colony (Fig. 3, A and B; Valerius et al., 2007). We first expressed the full-length S. cerevisiae CPC2 gene in the cpc2 mutant using the S. cerevisiae expression vector p424MET25 (Mumberg et al., 1994) and observed the restoration of pseudohyphae growth (Fig. 3C). With this validated system, we found that when any of the three Arabidopsis RACK1 genes were expressed in the S. cerevisiae cpc2 diploid mutant background, the transformant regained the ability to produce the filament-like structures (pseudohyphae; Fig. 3, D–F). These results demonstrated that the Arabidopsis RACK1 genes are functionally equivalent to the S. cerevisiae CPC2/RACK1. In an earlier study, Gerbasi et al. (2004) demonstrated that the mammalian RACK1 is also a functional ortholog of the S. cerevisiae CPC2 gene. In agreement with these genetic data, both the amino acid sequence (Chen et al., 2006) and crystal structure (Ullah et al., 2008) of RACK1 are also highly conserved in different eukaryotic organisms

Feature

The additional Pumilio protein (At5g64490; APUM26, predicted by Tam et al. (2010) was excluded from our analysis as it lacks a typical Pumilio homology domain or Puf domain; instead it contains an Armadillo-type fold, which is structurally similar to the Pumilio homology domain

Feature

the amino acid sequence corresponding to the basic region in ... PAR2 was clearly acidic in both proteins, this region was referred to as the acidic (A) domain (Figure S1b). No conserved motifs were found in the N-terminal (N) or C-terminal (C) regions

Feature

PAR1 ... were classified as atypical bHLH proteins because they show homology to this family of transcription factors only within the HLH (here referred to as H) region (Roig-Villanova et al., 2007). In particular they lack the H/K9-E13-R17 motif characterized as critical for proper DNA binding to the G-box sequence

Feature

PAR2 were classified as atypical bHLH proteins because they show homology to this family of transcription factors only within the HLH (here referred to as H) region (Roig-Villanova et al., 2007). In particular they lack the H/K9-E13-R17 motif characterized as critical for proper DNA binding to the G-box sequence

Feature

two truncated versions of HFR1 fused to the GFP reporter gene (G-BH and G-H) were generated (Figure S4) and overexpressed in transgenic plants under the 35S promoter (P35S:G-BH and P35S:G-H lines) (Figure 6a). The BH fragment that we overexpressed in this work comprises the same HFR1 residues of the CT161 construct generated by other authors, whose truncated gene product is more stable than the full-length HFR1 protein in the dark (Yang et al., 2005). The H fragment of the HFR1 gene overexpressed was analogous to the PAR1 HC construct described in Figure 1(a), because it contains the HLH and the adjacent C-terminal region. The GFP fluorescence indicated that both truncated proteins were expressed and targeted to the nucleus of plant cells (Figure 6b). As controls, untransformed plants (wt) and a transgenic line overexpressing an epitope-tagged version of HFR1 (P35S:HFR1-HA) were used (Duek et al., 2004). As an estimate of the biological activity of these HFR1 derivatives, we analyzed the response of hypocotyls to W + FR. Both P35S:G-BH and P35S:G-H seedlings displayed significantly short hypocotyls when grown under W and a significantly reduced response to W + FR compared with the untransformed wt control. The decreased hypocotyl elongation phenotype was stronger than in P35S:HFR1-HA lines (particularly in response to simulated shade) (Figure 6c). In all three cases, two-way anova tests indicated a significant interaction (P

Feature

our results indicate that PAR1 does not bind to the promoter of its target genes either directly or indirectly (by association with other transcription factors). Instead, PAR1 might regulate gene expression by dimerizing with other bHLH proteins

Feature

the truncated PAR1 versions tested only those containing the HLH domain (required for protein–protein interactions) and the adjacent C-terminal region have biological activity in the regulation of the SAS responses. By contrast, the N-terminal and acidic domains are not required for full (wild-type) PAR1 function in vivo, although the N-terminal region provides a functional NLS

Feature

the amino acid sequence corresponding to the basic region in PAR1 ... was clearly acidic in both proteins, this region was referred to as the acidic (A) domain (Figure S1b). No conserved motifs were found in the N-terminal (N) or C-terminal (C) regions

Feature

Previously it was shown that conserved residues in the HLH domain of PAR1 are required for homodimerization in a Y2H assay. In particular, PAR1L66E (a derivative of PAR1 containing Leu66 mutated to Glu) (Figure S1b) displayed no interaction in the Y2H growth assay (Carretero-Paulet et al., 2010). To study its biological activity, the mutated form fused to GFP was overexpressed in transgenic plants (P35S:PAR1L66E-G lines). At least two independent transgenic plants showing high levels of transgene expression were selected. The GFP fluorescence confirmed that the mutated PAR1L66E-G protein was produced and localized in the nucleus (Figure 5b). As shown in Figure 5(c,d), overexpression of PAR1L66E-G lacked any biological activity, based on the hypocotyl elongation in response to simulated shade and other morphological phenotypes, as well as on the expression of At5g57780, a marker gene of increased PAR1 activity (Figure 2). Altogether, these results confirm that dimerization via the HLH domain is essential for PAR1 activity in plant cells

Feature

To further define BES1 in vivo binding sites, we performed de novo motif discovery of the BR-regulated BES1 targets using the cosmo algorithm (Bembom et al., 2007) and found that while E-boxes are present in both BR-repressed and BR-induced genes, BRRE is more dominant in BR-repressed genes (Figure 3c,d). We performed gel mobility shift assay with labeled DNA probes containing BRRE as well as CACGTG and CACTTG E-boxes (Figure S2). BES1 can bind to both BRRE and E-boxes (CACGTG and CACTTG). The stronger binding of BES1 to BRRE element than to E-boxes is likely due to the fact that BES1 needs a heterodimer partner to bind E-box more efficiently

Feature

BES1 tended to bind to DNA sequences near the TSS (Figure 3a,b). In all BES1 targets (Figure 3a) and in the BR-regulated BES1 targets (Figure 3b), the highest frequencies of interval occurrence appeared to be around the TSS (–500–500) with a slight preference to the promoter side. So we conclude that BES1 tends to bind to DNA elements in close proximity to the TSS to regulate gene expression

Feature

The phylogenetic tree shows that the ASF1 proteins in the green lineage clearly distant from the animal homologues, with the algae and fern ASF1 proteins more closely related with the yeast homologues (Figure 1a). In general, lower eukaryotic species including yeast, algae, fern and Drosophila, each possess a single ASF1, whereas higher plants and mammals each species contains two ASF1 isoforms. Remarkably, the moss Physcomitrella patens contains four isoforms, and the two isoforms in the same species of higher plants are closely related, together suggesting that ASF1 multiplication/duplication had occurred relatively recently during evolution

Feature

The phylogenetic tree shows that the ASF1 proteins in the green lineage clearly distant from the animal homologues, with the algae and fern ASF1 proteins more closely related with the yeast homologues (Figure 1a). In general, lower eukaryotic species including yeast, algae, fern and Drosophila, each possess a single ASF1, whereas higher plants and mammals each species contains two ASF1 isoforms. Remarkably, the moss Physcomitrella patens contains four isoforms, and the two isoforms in the same species of higher plants are closely related, together suggesting that ASF1 multiplication/duplication had occurred relatively recently during evolution

Feature

TBLASTN search (Altschul et al., 1997) with the human PEP motif on the Arabidopsis thaliana and rice genomes identified a single gene encoding a protein with a PEP motif in each species (Figure 1a). Using the discovery motif MEME/MAST (Bailey and Elkan, 1994), we identified a second conserved motif in the C terminal extremity of plant putative NUFIP, human NUFIP and yeast Rsa1p (Figure 1a). This motif contains the CRM1-dependent nuclear export signal (NES) described in human NUFIP ... the predicted NUFIP homolog is encoded by gene At5g18440

Feature

The coding sequence of AtNUFIP gene was based on a partial cDNA. To eliminate any ambiguity, we mapped the 5' and 3' ends of AtNUFIP mRNA by RLM RACE. This mapping revealed two types of transcripts that differed by a 96 nucleotide intron retained on the 5'UTR (Figure 2a). Otherwise both transcripts have identical ORFs and 3'UTRs. Semi-quantitative RT-PCR on total RNA from wild type seedlings with primers flanking the retained intron (Figure 2a) confirmed that both transcripts are expressed at similar levels

Feature

To further characterize the eukaryotic NUFIP family, we extended the previous analysis to several species, including protist and green unicellular algae (Figure 1b). In addition to PEP, the NES motif is conserved in most species, except in a few lower eukaryotes (Figure 1b). Alignment of this motif shows high conservation of hydrophobic residues that are important for nuclear export ... At5g18440

Feature

To define the promoter region responsible for the upregulation of gene expression by VND7, we carried out transient reporter assays using the XCP1 promoter sequence. We constructed reporter plasmids by linking various lengths of XCP1 promoter sequences to a minimal CaMV 35S promoter driving the firefly luciferase gene (Figure 4a); the VND7 gene driven by the CaMV 35S promoter was used as an effector plasmid. When the region from -705 to -79 bp of the XCP1 promoter was used as the reporter, strong luciferase activity was detected, as reported previously (Figure 4b; Yamaguchi et al., 2010b). A 5' deletion series of XCP1 promoter (proB-F; Figure 4a) indicated that the luciferase activity obviously dropped in the proF construct (from -173 to -79 bp), suggesting that 98 bp of the XCP1 promoter fragment, corresponding to the region from -271 to -174 bp, includes the crucial region for VND7-inducible XCP1 expression. Sequential 5' deletion analysis revealed that a 38-bp fragment of the XCP1 promoter (from -211 to -174 bp) is necessary for XCP1 induction by VND7 ... To investigate the possibility that a promoter region other than the 38-bp region described above (from -211 to -174 bp) contributes to the induction by VND7, we performed 3' deletion of proE (from -271 to -79 bp) and proI (from -211 to -79 bp), to make proK (from -271 to -173 bp) and proL (from -211 to -173 bp), respectively. Interestingly, the expression levels of luciferase driven by proK and proL were lower than when driven by proF (Figure 4d). Thus, the proL fragment is not sufficient for the induction of gene expression by VND7. To determine an additional responsible region between -173 and -79 bp, we prepared a 3' deletion series of the XCP1 promoter (proN–proQ; Figure 4a). A decrease of luciferase activity was observed in the cases of proN, proP and proQ; in particular, proP and proQ lacked almost all luciferase activity, as shown for proL (Figure 4e). These results show the importance of the upstream region starting at -96 bp for VND7-inducible gene expression. We conclude that the region of the XCP1 promoter between residues -211 and -96 is necessary and sufficient for gene expression induced by VND7

Feature

The BUD23 deletion line (bud23Δ) of budding yeast has been reported to exhibit severely impaired growth (Niewmierzycka and Clarke, 1999). We examined whether the RID2 gene could complement this growth defect of bud23Δ. To this end, GAL1p::RID2, a chimeric construct consisting of the GAL1 promoter of budding yeast and the RID2 gene, was introduced into bud23Δ, and RID2 was forcibly expressed by culture in the presence of d-galactose. The expression of RID2 under the GAL1 promoter was checked by RT-PCR (Figure 7b). Expression of the RID2 gene did not alleviate the growth defect of bud23Δ, whereas expression of BUD23 in the same system did so (Figure 7a). This result showed that RID2 cannot act as a substitute for BUD23, despite their significant similarity in sequence

Feature

AtHsp70-14 (At1g79930) and AtHsp70-15 (At1g79920) are localized next to each other on the same strand of chromosome 1. According to recent annotations by the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org), both genes encode proteins with 831 amino acids, but, based on published ESTs, two slightly shorter splicing variants may also be expressed. At the nucleotide level, the isoforms show sequence identity of 69% in the untranslated 5′ and 3′ regions and 96% in the coding region. At the protein level, the isoforms are 97% identical

Feature

AtHsp70-14 (At1g79930) and AtHsp70-15 (At1g79920) are localized next to each other on the same strand of chromosome 1. According to recent annotations by the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org), both genes encode proteins with 831 amino acids, but, based on published ESTs, two slightly shorter splicing variants may also be expressed. At the nucleotide level, the isoforms show sequence identity of 69% in the untranslated 5′ and 3′ regions and 96% in the coding region. At the protein level, the isoforms are 97% identical

Feature

As OSR1 shares a conserved domain with ARGOS and ARL, which comprises an identical LPPLPPPP motif and two putative transmembrane helices (Fig. 5a), we named it the Organ Size Related (OSR) domain. It is likely that the OSR domain is responsible for the function of the three members of the OSR family. To test whether this is the case, we generated transgenic plants overexpressing different truncated OSR1 coding regions and examined their final leaf sizes. As shown in Fig. 5(b,c), transgenic plants harbouring a transgene that encodes a truncated OSR1 protein with an intact OSR domain or the OSR domain alone still exhibited enlarged organs, as did 35S-OSR1 plants. By contrast, overexpression of a transgene with a disrupted OSR domain could not recapitulate the organ phenotype of 35S-OSR1 plants. Consistently, overexpression of the OSR domain in ARGOS and ARL also resulted in the organ phenotypes of 35S-ARGOS and 35S-ARL, respectively (Fig. 5b,c). These results demonstrate that the OSR domain is essential and sufficient for promoting organ growth

Feature

As OSR1 shares a conserved domain with ARGOS and ARL, which comprises an identical LPPLPPPP motif and two putative transmembrane helices (Fig. 5a), we named it the Organ Size Related (OSR) domain. It is likely that the OSR domain is responsible for the function of the three members of the OSR family. To test whether this is the case, we generated transgenic plants overexpressing different truncated OSR1 coding regions and examined their final leaf sizes. As shown in Fig. 5(b,c), transgenic plants harbouring a transgene that encodes a truncated OSR1 protein with an intact OSR domain or the OSR domain alone still exhibited enlarged organs, as did 35S-OSR1 plants. By contrast, overexpression of a transgene with a disrupted OSR domain could not recapitulate the organ phenotype of 35S-OSR1 plants. Consistently, overexpression of the OSR domain in ARGOS and ARL also resulted in the organ phenotypes of 35S-ARGOS and 35S-ARL, respectively (Fig. 5b,c). These results demonstrate that the OSR domain is essential and sufficient for promoting organ growth

Feature

As OSR1 shares a conserved domain with ARGOS and ARL, which comprises an identical LPPLPPPP motif and two putative transmembrane helices (Fig. 5a), we named it the Organ Size Related (OSR) domain. It is likely that the OSR domain is responsible for the function of the three members of the OSR family. To test whether this is the case, we generated transgenic plants overexpressing different truncated OSR1 coding regions and examined their final leaf sizes. As shown in Fig. 5(b,c), transgenic plants harbouring a transgene that encodes a truncated OSR1 protein with an intact OSR domain or the OSR domain alone still exhibited enlarged organs, as did 35S-OSR1 plants. By contrast, overexpression of a transgene with a disrupted OSR domain could not recapitulate the organ phenotype of 35S-OSR1 plants. Consistently, overexpression of the OSR domain in ARGOS and ARL also resulted in the organ phenotypes of 35S-ARGOS and 35S-ARL, respectively (Fig. 5b,c). These results demonstrate that the OSR domain is essential and sufficient for promoting organ growth

Feature

the Arabidopsis genome encodes two EUI-like (EL) P450s, CYP714A1 and CYP714A2 (Zhu et al., 2006), referred to hereafter as ELA1 and ELA2, respectively. The two Arabidopsis proteins share 72.5% identity with each other, and 38% identity with EUI, and contain all conserved amino acids for heme binding, oxygen binding, and activation, and the ERR (Glu–Arg–Arg) triad motif known to be present in all cytochrome P450 proteins (Figure S1). Interestingly, the two ELA genes are located next to each other with only a 769-bp interval with a retrotransposon next to the ELA1 promoter, suggesting that they might be generated from a simple duplication

Feature

the Arabidopsis genome encodes two EUI-like (EL) P450s, CYP714A1 and CYP714A2 (Zhu et al., 2006), referred to hereafter as ELA1 and ELA2, respectively. The two Arabidopsis proteins share 72.5% identity with each other, and 38% identity with EUI, and contain all conserved amino acids for heme binding, oxygen binding, and activation, and the ERR (Glu–Arg–Arg) triad motif known to be present in all cytochrome P450 proteins (Figure S1). Interestingly, the two ELA genes are located next to each other with only a 769-bp interval with a retrotransposon next to the ELA1 promoter, suggesting that they might be generated from a simple duplication

Feature

We first tested the RNA-binding properties of these enzymes by UV crosslinking. As shown in Figure 6(b), the WT enzyme displayed a dissociation constant (Kd) of 22 nm, in the range of the 11 and 16.5 nm values for the E. coli and human mitochondrial enzymes (Portnoy et al., 2008). Competition with polynucleotides showed that as previously reported for spinach cpPNPase (Yehudai-Resheff et al., 2003), the Arabidopsis enzyme has a higher affinity for poly(U) and poly(A) than for poly(G) and poly(C) (data not shown). When the mutant enzymes were tested, P184L did not differ from the WT, R176S–P184L had a Kd value of 60 nm, and the G596R enzyme had no detectable RNA binding activity (Figure 6b

Feature

While the WT enzyme had robust polyadenylation and degradation activities (Figure 7a), G596R lacked both, consistent with its failure to detectably bind RNA in vitro (Figure 7b). P184L had reduced activity in both cases, as evidenced by the requirement for a higher Pi concentration to stimulate degradation, and lower production of poly(A) tails over time (Figure 7c). When the R176S–P184L double mutant was checked it had neither activity, suggesting that its weak RNA binding ability was insufficient to support catalysis under our experimental conditions

Feature

The significant effects of amino acid substitutions in the first core domain were somewhat unexpected, as it does not harbor the phosphorolytic site. In addition, while the intermediate catalytic activity of P184L was consistent with the RNA phenotypes observed by RNA gel blot analysis, the fact that the enzyme appeared to have normal RNA binding was counterintuitive

Feature

The presence of the C-terminal Cys-rich domain makes AGG3 quite unique and different from all other Gγ subunits studied so far. It is therefore important to determine whether the C-terminal domain is essential for the correct function of the AGG3 protein. For that purpose we performed complementation studies in the agg3-3 background using two truncated AGG3 proteins lacking the C-terminal region. Truncations as well as the full-length complementation constructs were amplified from genomic DNA and contain 2 kb of the native promoter as well as all four introns present in the native gene. The first truncation construct (trunc1) contained the entire γ-domain (residues 1–135) including a CaaX motif present at the end of the AGG1/2 alignment (residues 132–135; Figure 1a). The second truncation construct (trunc2) also contained the γ domain (residues 1–129), but differed from trunc1 in that we added the CaaX motif present at the C-terminus of the native gene (residues 246–251). Importantly, both truncated AGG3 constructs lack the Cys-rich region but contain a CaaX isoprenylation motif, so aberrant subcellular localization was not expected to be an issue. Five independent homozygous lines were analysed for each of the two AGG3 truncation constructs, of which we show two representative lines for simplicity. We measured silique length and width, flower and hypocotyl length, mature plant height, and seed germination in 2 μm ABA (uniform germination on standard 0.5 × MS plates is shown in Figure S2i). Complementation lines containing the full-length AGG3 construct showed statistically significant differences to the agg3-3 line, although several of the phenotypes were not fully restored to wild-type levels (Figure S3e–j). In contrast, lines containing either of the truncated AGG3 constructs were indistinguishable from the agg3-3 line and unable to confer even partial complementation. Therefore without the Cys-rich domain, the truncated AGG3 constructs were not able to provide functional complementation in the agg3-3 mutant background, underscoring the importance of the Cys-rich domain for the function of the AGG3 protein

Feature

BLAST searches using AGG2 as a query identified an additional unknown protein (over twice as large as AGG1 and AGG2), encoded by the Arabidopsis gene At5g20635 (GenBank locus AAT85756), with a relatively low level of homology (score 40.8 and expect value 3 × 10−4 over a 55-amino-acid region). Alignment of AGG1, AGG2 and AAT85756 showed that homology to the two known Arabidopsis γ-subunits resides in the N-terminal region of AAT85756, while the C-terminal region is extremely rich in cysteine residues, with 44 Cys in the last 128 residues

Feature

The strength and nature of the interaction was further studied by performing quantitative Y2H β-galactosidase activity assays using AGB1 and several N- and C-terminal AGG3 deletions (Figure 2a,b). Full-length AGG3 showed a strong affinity for AGB1 (compared to the standard p53/SV40 positive control; Li and Fields, 1993), and the γ-domain (AGG31−135) showed higher affinity for AGB1 than either AGG1 or AGG2. Conversely, the C-terminal Cys-rich domain of AGG3 (AGG3136–251) displayed no affinity for AGB1. Residues 1–78 of the AGG3 γ-domain, containing the coiled-coil domain, were not sufficient to bind AGB1; however, their removal did significantly decrease the signal from the β-galactosidase reporter gene (see AGG379−251 in Figure 2b; P = 0.0148 when compared to full-length AGG3). This suggests that the AGB1/AGG3 interaction is not due to non-specific binding of the coiled-coil domains, rather the coiled-coil interaction enhances the strength of the binding. Comparison of AGG31−78 and AGG31−99 indicates that the region containing residues 78–99 is important for binding to the β-subunit. This region most notably contains the highly conserved DPLL/I motif (box 2 in Figure 1a) which has been identified as an important contact patch between β- and γ-subunits (Temple and Jones, 2007

Feature

At5g20635 contains an open reading frame of 753 bp encoding a theoretical 251-amino-acid protein with a predicted molecular weight of 27.2 kDa. AAT85756 possesses several important features that qualify it as a putative γ-subunit. Six out of the eight AGG1 residues identified by Temple and Jones (2007) as being important for contact with AGB1 are conserved (L37, E40, S51, D66, P67 and L68), one is conservatively substituted (L69), and only N77 is not conserved in AAT85756 (highlighted in Figure 1a and Figure S1 in Supporting Information for homologues). Secondly, AAT85756 is predicted to contain an N-terminal coiled-coil domain (box 1 in Figure 1a), an important structural characteristic of γ-subunits providing strength to β/γ dimerization (Pellegrino et al., 1997; McCudden et al., 2005). Thirdly, the positions of the first three introns in AGG1, AGG2 and At5g20635 are identical (asterisks in Figure 1a), suggesting that At5g20635 may be the result of an ancient gene duplication event. Fourthly, AAT85756 contains a C-terminal isoprenylation (CaaX) motif (box 3 in Figure 1a), an important element conserved within G-protein γ-subunits (Simonds et al., 1991; Chakravorty and Botella, 2007). Finally, using Phyre, a software package that predicts the most similar available tertiary structures to a query using primary sequence information, the only hits with significant homology to AAT85756 (40–55% estimated precision), were γ-subunit structures from the heterotrimeric G protein (with no other hits over 20%; http://www.sbg.bio.ic.ac.uk/~phyre/; Kelley and Sternberg, 2009). We therefore decided to explore the possibility that AAT85756 might function as an unconventional G-protein γ-subunit, and tentatively named it AGG3

Feature

The interaction between the N-terminal fragment of AGG3 (residues 1–112), containing the entire γ-domain, and AGB1 was also confirmed using in vitro binding assays. [35S]-Methionine-labelled AGG31−112 was found to bind an immobilized glutathione S-transferase (GST)–AGB1 fusion protein, but not GST

Feature

we searched for putative orthologues of the yeast DExH box RNA helicase Mtr4p, a known nuclear exosome co-factor and component of the polyadenylating TRAMP complex. Four members of the large family of Arabidopsis DExH box RNA helicases belong to the sub-family of Ski2p/Mtr4p-like RNA helicases ... The uncharacterized protein encoded by At3g46960 has the best similarity score with the cytoplasmic exosome co-factor Ski2p

Feature

we searched for putative orthologues of the yeast DExH box RNA helicase Mtr4p, a known nuclear exosome co-factor and component of the polyadenylating TRAMP complex. Four members of the large family of Arabidopsis DExH box RNA helicases belong to the sub-family of Ski2p/Mtr4p-like RNA helicases (Figure S1). The cytoplasmic protein ISE2 (At1g70070), the least conserved member of the sub-family, is localized in cytoplasmic granules, and is involved in post-transcriptional gene silencing and the development of plasmodesmata (Kobayashi et al., 2007)

Feature

To study the role of the STM-binding sites on CUC1 transcription, we turned to reporters ... We analyzed the transcription of a wild-type reporter and two mutated versions where one or both STM-binding sites were removed. Mutations in the putative STM-binding sites quantitatively decreased its expression levels during vegetative development more than 2-fold by assaying seven independent transgenic lines for each construct

Feature

Next, we searched for potential STM regulatory motifs by analyzing the promoters of genes up-regulated in the microarray data, as described previously (Schommer et al., 2008). We only found a potential candidate box when we analyzed genes induced at least 5-fold by STM-VP16, GTCACT (P = 0.06; Supplemental Table S5). Even though the enrichment of this site was not particularly high, it suggestively overlapped with the preferred binding site of STM, which has already been investigated in vitro and was found to be CTGTCA (Krusell et al., 1997; Smith et al., 2002; Viola and Gonzalez, 2006). These sequences share the minimal sequence recognized by KNOX homeodomains, a GTCA core (for review, see Hake et al., 2004).

Feature

Feature

When the sequence of the FYF protein was analyzed, a conserved IDLNL sequence, similar to the EAR motif for the Class II ERF repressors (Ohta et al., 2001), was identified in the C-terminal region of FYF (Figures 5a and S4). The presence of this sequence indicated that the FYF gene might encode a transcriptional repressor

Feature

To analyze the underglycosylation defect of alg10-1 in more detail we performed SDS-PAGE and immunoblotting using antibodies specific for different glycoproteins. Previous studies have analyzed the mobility of the ER-retained glycoprotein protein disulfide isomerase (PDI) to monitor underglycosylation defects in plants (Hoeberichts et al., 2008; Kajiura et al., 2010; Lerouxel et al., 2005; Zhang et al., 2009). In the alg10-1 mutant three PDI forms were detectable while in the wild type a single PDI form was present (Figure 7a). Upon digestion with endoglycosidase H (Endo H) or peptide: N-glycosidase F (PNGase F) the three bands shifted to a band that migrated at the same position as the de-glycosylated wild-type protein, showing that PDI is underglycosylated in alg10-1 (Figure 7b). Importantly, analysis of PDI forms present in different underglycosylation mutants revealed that ALG10 loss-of-function results in a more severe defect than observed for alg3 and stt3a-2 mutants as the underglycosylated PDI forms were more abundant in alg10-1

Feature

To identify the putative Arabidopsis α1,2-glucosyltransferase that catalyzes the final glucosylation step during the biosynthesis of the dolichol-linked oligosaccharide precursor (Figure 1) we used the amino acid sequence of the Saccharomyces cerevisiae ALG10 (Burda and Aebi, 1998) and performed a BLASTP search in the A. thaliana protein database. As a result of this search we identified a single protein encoded by the At5g02410 gene. This protein has been annotated to the glycosyltransferase family GT59 in the Carbohydrate-Active-enZYmes database (CAZY; http://www.cazy.org/), which contains inverting enzymes that transfer glucose residues from dolichol-P-glucose in α1,2-linkage to Glc2Man9GlcNAc2-PP-Dol, the ultimate step in the assembly of the oligosaccharide precursor

Feature

To determine whether ALG10 is a functional ortholog of the yeast ALG10 glycosyltransferase we expressed the full-length Arabidopsis ALG10 open reading frame under the control of a constitutive promoter in the S. cerevisiae Δalg10 knockout strain and tested for complementation of the mutant phenotype. In yeast, ALG10 deficiency results in severe underglycosylation of N-linked glycoproteins because the oligosaccharyltransferase transfers incompletely assembled oligosaccharides with reduced efficiency (Burda and Aebi, 1998). The hypoglycosylation of proteins can be monitored by immunoblotting using antibodies against the vacuolar protease carboxypeptidase Y (CPY). Yeast CPY carries four N-linked glycans and in the Δalg10 strain two faster-migrating CPY-forms with a reduced number of N-glycans are detected. As shown in Figure 3(a), expression of Arabidopsis ALG10 in Δalg10 resulted in a reduced number of faster-migrating CPY-forms indicating partial rescue of the CPY underglycosylation defect

Feature

To obtain further evidence for the functionality of Arabidopsis ALG10 we analyzed the restoration of the lipid-linked oligosaccharide defect of the S. cerevisiae Δalg10 strain. The lipid-linked oligosaccharides were isolated from microsomal fractions, hydrolyzed and analyzed by liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS) analysis. In contrast to wild-type cells, which accumulated a peak corresponding to the fully-assembled Glc3Man9GlcNAc2 precursor, the Δalg10 mutant displayed a major peak representing Glc2Man9GlcNAc2 (Figure 3b) and smaller amounts of Glc1Man9GlcNAc2 and Man9GlcNAc2 (data not shown) (Burda and Aebi, 1998). The Δalg10 yeast strain expressing Arabidopsis ALG10 accumulated a peak that co-eluted with Glc3Man9GlcNAc2. These data show that Arabidopsis ALG10 can restore the lipid-linked oligosaccharide biosynthesis defect of the Δalg10 mutant yeast strain, indicating that it is the corresponding plant α1,2-glucosyltransferase

Feature

We amplified the whole open reading frame including additional 5′- and 3′-untranslated regions of the Arabidopsis ALG10 from leaf cDNA. The sequence of the open reading frame was identical to the annotated one from the TAIR database and encodes a protein of 509 amino acid residues. The Arabidopsis ALG10 has 26% identity (44% similarity) to the S. cerevisiae ALG10 amino acid sequence (Figure S1 in Supporting Information). It contains three putative N-glycosylation sites and bioinformatic analysis (Plant Protein Membrane Database, http://aramemnon.botanik.uni-koeln.de/) predicts the presence of 12 transmembrane helices (Figure S1) with both ends facing the cytosol as has been suggested for yeast ALG10 (Oriol et al., 2002). Consistent with yeast ALG10, the Arabidopsis homolog does not contain any C-terminal dilysine motif, which can typically be found in other ER-located yeast and Arabidopsis ALG proteins (Oriol et al., 2002; Henquet et al., 2008; Hong et al., 2009; Kajiura et al., 2010), and acts as a Golgi-to-ER-retrieval signal for these proteins

Feature

The Arabidopsis genome contains one gene (At2g18290) putatively encoding APC10 (Eloy et al., 2006). To verify whether this APC10 gene effectively encodes a functional protein, the coding sequence was inserted into a yeast expression vector and transformed into a temperature-sensitive apc10ts fission yeast (apc10-27) strain (Kominami et al., 1998). The expression of the Arabidopsis APC10 was able to rescue the apc10ts phenotype at the restrictive temperature of 35°C, while the negative control containing the antisense construct was unable to grow under the same conditions

Feature

ARF2 contains the N-terminal DNA-binding domain that targets the AuxREs without the help of a middle or C-terminal part (the middle region for transcriptional activation or repression, and the C-terminal dimerization domain

Feature

HB33 in Arabidopsis belongs to a zinc finger-homeodomain (ZF-HD) subfamily containing 14 members that can dimerize with each other in a yeast two-hybrid assay [30]. Most proteins in this family do not have an intrinsic activation domain and might need to interact with other factors for transcriptional activation [30

Feature

The MDO1 gene encodes a 127 amino acid protein, the primary sequence of which contains no known functional domain, including a subcellular localization signal. Interestingly, the MDO1 sequence is highly conserved in a wide variety of land plants, including a moss, but not in other organisms, such as animals. The mdo1-1 mutation is located within a five amino acid tract (FIGEL) that is completely conserved in all known homologs

Hashimura Y, Ueguchi C - The Arabidopsis MERISTEM DISORGANIZATION 1 gene is required for the maintenance of stem cells through the reduction of DNA damage

Feature

Analysis of RUG2 with smart (http://smart.embl-heidelberg.de) revealed a modular architecture with 10 mTERF motif repeats, each of which is about 31 amino acids in length (Figure 4). Interestingly, a proline residue is highly conserved at position 8 of every mTERF motif

Feature

The amino acid sequence of the MAN gene was compared with other ANs (Figure 1). The MAN protein had a conserved 2-Hacid_DH motif lacking a catalytic triad and long C-terminus, similar to other AN proteins. In the MAN protein, the amino acid sequence of the LxCxE/D motif found in all plant AN proteins was LxCxD, similar to LgAN from Dahurian larch (Lin et al., 2008). Initially, it was reported that the LxCxE motif was responsible for binding to retinoblastoma protein in animals. It was then reported that mutant E1A with the LxCxD mutation still binds to Rb (Corbeil et al., 1994) and that human cytomegalovirus pp71 protein employs an LxCxD motif to attack the Rb pathway (Kalejta and Shenk, 2003). Therefore, E to D substitution may have no effect on the role of the motif, even though the function of this LxCxE/D motif in the AN proteins was not determined. On the other hand, the putative NLS sequence KKRH, reported previously for AN (Folkers et al., 2002; Kim et al., 2002) and IAN (Cho et al., 2005), was changed to the sequence KKRA in MAN. Moreover, a putative PEST motif reported to be in AN (Folkers et al., 2002) was not detected by the PEST-find program. The latter two motifs are suspected to be collapsed in MAN. We transformed Arabidopsis an-1 with 35S-drived MAN and examined whether it complements the an-1 phenotype, with reference to transgenic an-1 with 35S-derived authentic AN. Although the an-1 mutant plant expresses an-1 mutant protein that has an abnormal sequence in the C-terminal region (from a site 22 bp upstream of the termination codon in the 3′ sequence of the AN gene; Kim et al., 2002), we found that six amino acids of the C-terminal region is indispensable for homodimerization of the AN protein (Figure S1), enabling an-1 to be used as a null allele. RNAi lines for AN also confirmed that the observed an-1 phenotypes are indeed derived from loss of function of AN protein (data not shown). As a result of transformation, MAN was found to be completely functional in Arabidopsis (Figure 2 and Table 1), irrespective of the abovementioned differences in the motifs

Feature

Motifs found in AN are the putative nuclear localization signal (KKRH), a retinoblastoma (Rb)-binding motif (LxCxE/D), and a cell cycle-specific phosphorylation motif (IAMSD) (Figure 1). The above results from analyses of the liverwort homolog, however, suggested that the NLS sequence might be unnecessary for AN function, at least for leaf-shape and trichome-branching controls. To examine this, we used mutagenized lines whose known motifs were substituted as follows: KKRH to AAAA, LxCxE to RxRxG, and IAMSD to IAMAD, respectively, for the putative NLS, the Rb-binding motif, and the putative phosphorylation motif. Although it was reported that AN also has a PEST motif, mutagenized AN cDNA with substitution in the motif was not constructed in this experiment because of poor definition of the PEST motif. AN cDNAs with substitutions in these motifs were transformed into the an-1 mutant, and we examined whether they complemented the an-1 phenotype. For each construction of mutated AN, more than six lines of homozygous transformants were obtained. Phenotypes of these lines were checked for the leaf index and number of trichome branches. Although some lines did not recover the an-1 phenotype, seemingly because of low expression levels of transformed cDNAs, each construct had lines that recovered the an-1 phenotype to a certain extent, suggesting that all these constructions are functional

Feature

To test if the NAC domain is responsible for the nuclear localization of SND1 and to further assess the effect of the point mutation on NAC domain function, we constructed an NAC domain-YFP fusion consisting of 191 amino acids that included the entire NAC domain of SND1 (subdomains A–E) (Shen et al., 2009). The wild-type NAC domain-YFP was localized exclusively to the nucleus, whereas the NACm-YFP, which contains the point mutation, again showed both nuclear and cytoplasmic localization (Figure 4). These results indicate that the point mutation somehow affects the nuclear localization function of the NAC domain, but this may not be the main reason for the loss of function, as most of the signal is still in the nucleus

Feature

To investigate this phenomenon at the level of promoter binding, wild-type and mutant NAC domain polypeptides were expressed in Escherichia coli as fusions to maltose-binding protein (MBP), and the proteins were purified for the DNA binding assay (Figure S2). The same promoter fragment of MYB46 as used for the trans-activation assay was then used in EMSAs for the direct demonstration of promoter binding. The direct binding of SND1 to this fragment was confirmed by adding unlabeled competitor DNA (Figure 5b, lane 4). The T94K mutated SND1 protein, however, totally lost binding ability

Feature

To further narrow down the promoter region of SND1 needed for SND1 binding, we focused on the P2 fragment and synthesized nine short overlapping fragments encompassing this sequence (Figure 6d). In competition experiments, we found that P2-5 and P2-9 could compete with the biotin labeled full-length P2 probe (Figure 6e). The P2-5 fragment is shared by P2 and P3. To test if P2-5 is also responsible for the binding of SND1 to the P3 fragment, we checked for competition of P2-5 with a biotin-labeled P3 probe. P2-5 did indeed compete with P3, and was the only fragment that could do so (Figure S4), indicating that P2-5 contained the binding sequence for SND1 in the SND1 promoter. P2-9 is shorter than P2-5, and can also compete with the P2 fragment. To determine the critical nucleotides required for SND1 binding, we compared the P2-9 fragment with the published binding sequence of SND1 in the MYB46 promoter (Figure 7a) (Zhong et al., 2007). A consensus sequence, TATACXTTXXXXATGA, was found between the two. To check if all the nucleotides in the consensus sequence are critical we generated a series of mutants (M1–M5; Figure 7b), and tested these as competitors. Only M1 competed as strongly as the wild-type sequence, indicating that the first two nucleotides are not critical. We therefore conclude that the critical nucleotides required for SND1 binding are TACXTTXXXXATGA

Feature

Sequences 3 kb upstream of the translation start site of each annotated gene in the TAIR9 database (ftp://ftp.arabidopsis.org/home/tair/Sequences/blast_datasets/TAIR9_blastsets/database) were searched for the presence of the TACXTTXXXXATGA motif. This element is present in 647 promoters in the Arabidopsis genome (Table S2), including the promoter of MYB32, which is a known target for regulation by SND1

Feature

Arabidopsis AtSND1 is homologous to MtNST1, and has been shown to activate the secondary cell wall synthesis program. To check whether the T94K mutation may also cause the loss of function of AtSND1, we introduced the point mutation into AtSND1 by PCR and analyzed the trans-activation activity of the corresponding recombinant protein using a dual luciferase system. As reported previously (Zhong et al., 2006; Ko et al., 2009), the wild-type SND1 expression construct could activate the promoters of a number of cellulose, xylan and lignin biosynthesis genes. However, the T94K mutant could barely activate any of the promoters (Figure 3a,b), indicating that T94 is critical for SND1 function

Feature

The T94K mutation affects the subcellular localization and DNA binding activity of SND1 ... A large portion of the mutated AtSND1-GFP signal was localized to the nucleus, but signal was also observed in the cytoplasm

Feature

To test this hypothesis, we produced and characterized homozygous AtBT1::T-DNA mutants constitutively expressing ΔTP′-AtBT1 and ΔTP′′-AtBT1, which encode truncated forms of AtBT1 lacking eight and 17 amino acids, respectively, from the N-terminal extension that acts as a plastidic transit peptide (Figure 2a–c, Table 1, and Figures S1 and S3) (Bahaji et al., 2011). We also produced AtBT1::T-DNA mutants expressing ΔTP′-AtBT1 under the control of the AtBT1 promoter (AtBT1pro-ΔTP′-AtBT1) (Table 1 and Figures S1 and S4). To confirm that AtBT1 was specifically targeted to mitochondria in plants expressing ΔTP′-AtBT1, we also produced and characterized wild-type plants expressing ΔTP′-AtBT1-GFP. Because targeting sequences to mitochondria are located within the mature part of AtBT1 (Bahaji et al., 2011), it was impossible to produce homozygous AtBT1::T-DNA mutants expressing AtBT1 in the plastid only ... Fluorescence confocal microscopy studies of ΔTP′-AtBT1-GFP-expressing wild-type plants showed that GFP fluorescence distribution and motility patterns were identical to those of plants expressing a mitochondrial-targeting pre-sequence fused with GFP (MitTPr-GFP), and were clearly distinguishable from those of plants expressing the whole AtBT1 protein fused with GFP (Figure 3 and Figure S5 and movies S1-S3; see also Bahaji et al., 2011), confirming that AtBT1 in ΔTP′-AtBT1-, ΔTP′′-AtBT1- and AtBT1pro-ΔTP′-AtBT1-expressing cells is exclusively targeted to mitochondria. Most importantly, homozygous AtBT1::T-DNA mutants expressing ΔTP′-AtBT1, ΔTP′′-AtBT1 and AtBT1pro-ΔTP′-AtBT1 displayed a normal growth phenotype and produced fertile seeds (Figure 2d, e and Figures S3 and S4). As shown in Figure 4, ΔTP′-AtBT1 expression was still stable and complemented the aberrant growth and sterility phenotype of homozygous AtBT1::T-DNA mutants after several self-crosses and selection on hygromycin. The overall data thus show that (i) delivery of AtBT1 to mitochondria is enough to complement the aberrant growth and sterility phenotype of homozygous AtBT1::T-DNA mutants, and (ii) plastidic AtBT1 is not strictly required for normal development and fertility of the plant

Feature

We also tested whether four known co mutations, which cause missense mutations in the B-box domains (Robson et al., 2001), affect the interaction with AS1. All four CO variants attenuated the binding to AS1 in vitro (Figure S1), further supporting the notion that the B-box domains are important for the interaction with AS1

Feature

To further define the domain of interaction of CO with AS1, we tested the interaction of AS1 with various truncated CO proteins (Figure 1b). As expected, AS1 interacted with the truncated CO proteins that contain two B-box domains (Figure 1c). The truncated CO protein that possesses only the first B-box domain (designated B1) was not sufficient to bind to AS1 (Figure 1c). These results indicate that either the second B-box domain or both B-box domains may be involved in the interaction with AS1

Feature

Both NUS1 and AtNUS1 have seven exons, and the positions of introns at the amino acid level are fully conserved between these genes (Figures 1c, 8a and S1). The putative mature AtNUS1 protein (amino acid residues 82–462) is 63% identical and 93% similar to the corresponding region of NUS1. Our NCBI CDD searches suggested that AtNUS1 also carries a bacterial NusB-like domain at the C-terminus. We could not find any other Arabidopsis proteins with high sequence similarity and similar domain architecture, suggesting that NUS1 is present as a single copy in Arabidopsis as well as in rice

Feature

Structural analysis of ACD2 indicates possible roles for Glu154 and Asp291 in substrate binding and/or enzyme catalysis (Sugishima et al., 2009). Therefore, we tested whether the enzymatic activity and/or substrate binding is important for the cytoprotective role of ACD2. We generated a variant of ACD2 (ACD2**) in which both Glu154 (Glu154Ala; E154A) and Asp291 (Asp291His; D291H) residues were altered. Interestingly, RCC bound equally well to ACD2** and ACD2, as assessed by tryptophan fluorescence quenching (Figure 4a). In contrast, a coupled pheophorbide a oxygenase (PAO)/ACD2 enzyme assay using pheophorbide a as substrate with PAO isolated from bell pepper fruits and ACD2 variants indicated that ACD2** and the E154A variant completely lost their enzymatic activities, as no enzymatic product, pFCC, was produced ... To discern the functional consequences of a loss of enzyme activity but not RCC binding, we targeted ACD2** to acd2 chloroplasts (acd2/c-ACD2**) and mitochondria (acd2/m-ACD2**), respectively (Figure 5a). Targeting ACD2** either to chloroplasts or mitochondria did not alter the protoplast viability (Figure 5b) or the cell death initiation or progression relative to acd2 in any of the 72 plants analyzed from each targeted line. Mitochondrial H2O2 and 1O2 were also not altered in acd2/m-ACD2** or acd2/c-ACD2** compared with acd2 (Figure S6). Thus, the cytoprotective function of ACD2 is dependent on its catalytic activity

Feature

The predicted protein sequence of SAG113 has 34, 45 and 47% amino acid similarity to yeast PTC1 (Maeda et al., 1993), Arabidopsis ABI1, and Arabidopsis ABI2 proteins, respectively (Figure 5a). A phylogenetic tree also shows that it has a high similarity to ABI1, ABI2, and other PP2C group A subfamily proteins

Feature

To examine whether SAG113 has in vivo protein phosphatase activity, we performed complementation studies of a yeast mutant (Figure 5c). In yeast, the PTC1 gene encodes a functional PP2C, the disruption of which leads to a temperature-sensitive growth defect, i.e. the mutant yeast cells grow more slowly at 37°C than at 30°C (Maeda et al., 1993; Figure 5c). ABI2 and some other functional protein phosphatases have been reported to be able to complement the yeast temperature-sensitive mutant ptc1Δ (Leung et al., 1997; Hansen and Pilgrim, 1998; Kapranov et al., 1999). The SAG113 coding sequence was thus cloned into a yeast expression construct pWV3 under the ADH1 promoter to investigate whether it could also rescue the yeast ptc1Δ mutant. The construct pGL3216 containing the SAG113 was able to reverse the temperature-sensitive growth defect of ptc1Δ (Figure 5c), indicating that SAG113 protein was able to functionally compensate for the disrupted yeast PP2C, whereas the empty control vector pWV3 was unable to do so

Feature

The predicted protein sequence of SAG113 has 34, 45 and 47% amino acid similarity to yeast PTC1 (Maeda et al., 1993), Arabidopsis ABI1, and Arabidopsis ABI2 proteins, respectively (Figure 5a). A phylogenetic tree also shows that it has a high similarity to ABI1, ABI2, and other PP2C group A subfamily proteins

Feature

The predicted protein sequence of SAG113 has 34, 45 and 47% amino acid similarity to yeast PTC1 (Maeda et al., 1993), Arabidopsis ABI1, and Arabidopsis ABI2 proteins, respectively (Figure 5a). A phylogenetic tree also shows that it has a high similarity to ABI1, ABI2, and other PP2C group A subfamily proteins

Feature

To further determine whether LFY directly regulates the expression of AS2, we used chromatin immunoprecipitation (ChIP) to test whether LFY directly binds to the AS2 regulatory sequences in vivo. Based on LFY ChIP–chip data obtained by Winter et al. (2011), both primary LFY motifs, which contain critical nucleotides bound to a LFY homodimer, and secondary LFY motifs, which contribute to stage-specific LFY recruitment, were identified in the AS2 promoter, 5′ UTR, introns and exons

Feature

To further determine whether LFY directly regulates the expression of AS2, we used chromatin immunoprecipitation (ChIP) to test whether LFY directly binds to the AS2 regulatory sequences in vivo. Based on LFY ChIP–chip data obtained by Winter et al. (2011), both primary LFY motifs, which contain critical nucleotides bound to a LFY homodimer, and secondary LFY motifs, which contribute to stage-specific LFY recruitment, were identified in the AS2 promoter, 5′ UTR, introns and exons

Feature

The SLG1 gene contains a single exon with an open reading frame of 2550 bp that encodes a 95-kDa protein of 850 amino acid residues (Figure 4a). The encoded protein belongs to the E+ subclass of the Arabidopsis PPR protein family (PPR_5_2745209, Lurin et al., 2004). This PPR protein (SLG1) is predicted to have a mitochondrial targeting signal peptide at its N-terminus, 16 PLS repeats dispersed throughout the whole protein, and an E and E+ motif at its C-terminus (Figure 4a). Among these 16 PLS repeats are nine classic PPR motifs, five PPR-like L motifs, and two PPR-like S motifs

Feature

We directly sequenced all the editing sites of mitochondrial transcripts in the slg1 mutant. In the WT, 72% of the C nucleotides at position 250 of nad3 transcripts (nad3-250) were edited, but editing of nad3-250 was completely abolished in slg1 (Figure 6a). Meanwhile, the editing level of a nearby site, nad3-254, was unaffected in slg1. This indicated that the editing function of SLG1 was highly sequence-specific. The failure to edit the nad3-250 site converted the codon from TCT to CCT, resulting in a change from serine to proline

Feature

A BLAST search using the whole length of SLG1 as a query identified its close homologs in many plants species, including Vitis vinifera, Populus trichocarpa, Ricinus communis, Oryza sativa, and Sorghum bicolor (Figure 4b). SLG1 shares high similarities with these homologs throughout the whole protein sequence, except for the signal sequence for mitochondrial targeting. This result indicates that SLG1 proteins are highly conserved in higher plants

Feature

another RT-PCR analysis revealed an increase in RRC1 transcript size in the rrc1-1 mutant (Figure S3b,c), and we confirmed this result by sequencing the RRC1 mRNA; the altered alternative splicing was pinpointed to a region between exons 15 and 16 of RRC1

Feature

RRC1 was predicted to encode a 946-amino-acid protein, and the RRM is located between residues 180 and 256. At its C-terminus, we observed an amino acid sequence that is rich in serine/arginine/aspartic acid residues and contains one arginine/serine dipeptide. Within this region, we defined the C-terminal 102 residues as the RS domain of RRC1, which corresponds to that of human SR140

Feature

We reasoned that, because obvious developmental abnormalities were observed only in rrc1-4 but not for the other rrc1 alleles, the truncated RRC1 proteins that were functional enough to complement the pleiotropic defects should be expressed in rrc1-1, rrc1-2 and rrc1-3 (collectively hereafter called the ΔRS alleles). Indeed, determining the 3′-terminal sequence of wild-type and mutant RRC1 mRNAs by 3′ rapid amplification of cDNA ends (RACE) analysis revealed that the mutant mRNAs that coded for the truncated RRC1 proteins that lacked the RS domain were expressed by the ΔRS alleles (Figures 3a and S4). We then generated two different anti-RRC1 polyclonal antibodies, anti-RRC1-a and anti-RRC1-b, which recognize a region that spans amino acid residues 591–743 and the last 20 amino acid residues of the C-terminus of RRC1, respectively (Figure 3a). We investigated whether the truncated RRC1 proteins could be detected in mutants carrying the rrc1ΔRS alleles with these antibodies. Immunoblot analysis of the wild-type plants using anti-RRC1-a detected a major band of approximately 105 kDa. Since anti-RRC1-b detected a band of the same size, we concluded that this band represents full-length RRC1 (Figure 3b). However, in each rrc1ΔRS allele, anti-RRC1-a recognized a major band with a smaller molecular mass of approximately 100 kDa (for rrc1-1) or 85 kDa (for rrc1-2 and rrc1-3) (Figure 3b). These sizes were consistent with the expected mass of the deduced truncated RRC1 proteins (rrc1-1, 102.1 kDa and rrc1-2/rrc1-3, 90.6 kDa). Moreover, in rrc1-1 and rrc1-3 we were unable to detect any specific bands with anti-RRC1-b, which is an antibody that recognizes the RS domain of RRC1 (Figure 3b). Therefore, we concluded that the rrc1ΔRS alleles, which are defective only in phyB signaling, produce truncated RRC1 proteins that lack the RS domain. We noticed that both of the antibodies detected a faint band with the expected mass of full-length RRC1 in the rrc1-2 plants (Figure 3b). This was probably because the penultimate intron, which contained the T-DNA insertion, was spliced out in a fraction of the transcripts. These results suggest that the developmental defects that were observed in the null allele were complemented by the activity of these truncated RRC1 proteins. Thus, in the ΔRS alleles, the truncated RRC1 proteins that lack the RS domain retained the general function of RRC1 that is required for various developmental processes under normal conditions

Feature

Using the S. cerevisiae ERG10 sequence as the query, homology searches of the Arabidopsis genome revealed five genes encoding thiolases. Comparison of the five predicted amino acid sequences indicates that three genes (At1g04710, At2g33150 and At5g48880) encode type I thiolases, which are designated KAT1, KAT2 and KAT5

Feature

Using the S. cerevisiae ERG10 sequence as the query, homology searches of the Arabidopsis genome revealed five genes encoding thiolases. Comparison of the five predicted amino acid sequences indicates that three genes (At1g04710, At2g33150 and At5g48880) encode type I thiolases, which are designated KAT1, KAT2 and KAT5

Feature

In addition, each Arabidopsis AACT protein was recombinantly produced in Escherichia coli, and the kinetic parameters for each purified enzyme were determined (Figure S2). Both proteins are capable of catalyzing the production of acetoacetyl CoA from acetyl CoA, but, as indicated by the kcat constants for the two enzymes (503 ± 26 and 87 ± 5 min−1, respectively), AACT2 is a more efficient catalyst than AACT1 by a factor of six. The higher catalytic efficiency is primarily due to a higher Vmax value, and the Km is twofold lower for AACT2 (Figure S2

Feature

Two independent strategies were used to establish that the At5g47720 and At5g48230 genes encode AACT: (i) genetic complementation of the yeast erg10 mutant, and (ii) direct enzymological assay of the AACT activity supported by each recombinantly produced gene product. In yeast, AACT is essential for survival, as indicated by the fact that deletion of the AACT gene (ERG10) is lethal (Hiser et al., 1994). Therefore, a ΔERG10 heterozygous diploid S. cerevisiae strain was used to perform complementation experiments. Each Arabidopsis AACT open reading frame was expressed ectopically in the ΔERG10 heterozygous diploid strain using the galactose-inducible GAL1 promoter. The transformed diploid strain was sporulated and haploid cells recovered. Haploid ΔERG10 mutants expressing AACT1 or AACT2 grew on galactose-containing inductive medium (YPG), but not on non-inductive glucose-containing medium (YPD), indicating that both gene products complemented the erg10 mutation

Feature

Using the S. cerevisiae ERG10 sequence as the query, homology searches of the Arabidopsis genome revealed five genes encoding thiolases. Comparison of the five predicted amino acid sequences indicates ... At5g47720 and At5g48230 ... encode type II thiolases ... These two genes are located within 0.22 Mb of each other on chromosome 5, and are designated AACT1 and AACT2, respectively (Lange and Ghassemian, 2003); the two genes encode proteins that share approximately 80% sequence identity (Figure S1). Phylogenetic analyses indicated that the two Arabidopsis AACTs are closely related to cytosolic orthologs from rice, fungi (such as S. cerevisiae, Neurospora crassa and Aspergillus), and cytosolic and mitochondrial AACTs of Drosophila, mouse and humans (Pereto et al., 2005; Carrie et al., 2007

Feature

Two independent strategies were used to establish that the At5g47720 and At5g48230 genes encode AACT: (i) genetic complementation of the yeast erg10 mutant, and (ii) direct enzymological assay of the AACT activity supported by each recombinantly produced gene product. In yeast, AACT is essential for survival, as indicated by the fact that deletion of the AACT gene (ERG10) is lethal (Hiser et al., 1994). Therefore, a ΔERG10 heterozygous diploid S. cerevisiae strain was used to perform complementation experiments. Each Arabidopsis AACT open reading frame was expressed ectopically in the ΔERG10 heterozygous diploid strain using the galactose-inducible GAL1 promoter. The transformed diploid strain was sporulated and haploid cells recovered. Haploid ΔERG10 mutants expressing AACT1 or AACT2 grew on galactose-containing inductive medium (YPG), but not on non-inductive glucose-containing medium (YPD), indicating that both gene products complemented the erg10 mutation

Feature

Using the S. cerevisiae ERG10 sequence as the query, homology searches of the Arabidopsis genome revealed five genes encoding thiolases. Comparison of the five predicted amino acid sequences indicates that three genes (At1g04710, At2g33150 and At5g48880) encode type I thiolases, which are designated KAT1, KAT2 and KAT5

Feature

Using the S. cerevisiae ERG10 sequence as the query, homology searches of the Arabidopsis genome revealed five genes encoding thiolases. Comparison of the five predicted amino acid sequences indicates ... At5g47720 and At5g48230 ... encode type II thiolases ... These two genes are located within 0.22 Mb of each other on chromosome 5, and are designated AACT1 and AACT2, respectively (Lange and Ghassemian, 2003); the two genes encode proteins that share approximately 80% sequence identity (Figure S1). Phylogenetic analyses indicated that the two Arabidopsis AACTs are closely related to cytosolic orthologs from rice, fungi (such as S. cerevisiae, Neurospora crassa and Aspergillus), and cytosolic and mitochondrial AACTs of Drosophila, mouse and humans (Pereto et al., 2005; Carrie et al., 2007

Feature

The C-terminal tail of ACS4 contains a number of lysine residues, four of which (Lys424, -427, -452 and -453) are found in all or almost all type-II ACSs (Figures 1 and S1a in the Supporting Information). To determine if these lysine residues are involved in ACS4 degradation we investigated the effects of loss of all four lysine residues on ACS4 stability. A cell-free degradation assay was used to compare the turnover of His-Flag-ACS4 and His-Flag-ACS4(RR-RR) with Lys424, -427, -452 and -453 mutated to arginine (R). His-Flag-ACS4 and His-Flag-ACS4(RR-RR) recombinant proteins were incubated with extracts prepared from wild-type seedlings. Unexpectedly, the reduction in His-Flag-ACS4(RR-RR) levels occurred at a much faster rate than His-Flag-ACS4 (Figure S1b). These results are similar to those obtained for His-Flag-ACS7(K435R), where substituting a lysine residue for arginine in the C-terminal tail increased the proteasomal-dependent turnover of the protein (Figure 2b). These results suggest that these lysine residues in the ACS4 C-terminal tail are not utilized for ubiquitination but instead seem to be involved in stabilizing the protein

Feature

The covalent attachment of a polyubiquitin chain to a lysine residue on a target protein is a prerequisite for degradation by the 26S proteasome. ACS7 contains a single lysine residue (lys435) in its C-terminal tail (Figure 1). Therefore we investigated the possibility that lys435 was a site of ubiquitin attachment. Cell-free degradation assays were used to compare the stability of His-Flag-ACS7 to that of His-Flag-ACS7(K435R) which contains a lysine 435(K435) to arginine (R) mutation. If lys435 is used for ubiquitination, then loss of the lysine should retard degradation of ACS7. Unexpectedly, His-Flag-ACS7(K435R) was degraded much faster than His-Flag-ACS7 (Figure 2b). The degradation of His-Flag-ACS7(K435R) was blocked by MG132, suggesting that the increased turnover of the mutant ACS7 was carried out by the 26S proteasome (Figure 2b). These results suggest that lys435 is not utilized by the ubiquitination pathway. However, the fact that the mutation altered the rate of degradation suggests that the C-terminal tail lysine somehow influences the stability of ACS7

Feature

The SAUR19–24 genes of Arabidopsis are found in a tandem array on chromosome V and encode highly related (93–96% identity) proteins. Prior phylogenetic analysis revealed that SAUR19–24 form a unique clade in Arabidopsis (Jain et al., 2006). At 88–91 amino acids, members of the SAUR19–24 clade are among the smallest SAUR proteins. In general, larger SAURs have N- or C-terminal extensions, with their middle regions being most closely related to SAUR19–24 (Figure S1). As such, SAUR19–24 may represent a ‘minimal’ or ‘core’ SAUR sequence

Feature

The SAUR19–24 genes of Arabidopsis are found in a tandem array on chromosome V and encode highly related (93–96% identity) proteins. Prior phylogenetic analysis revealed that SAUR19–24 form a unique clade in Arabidopsis (Jain et al., 2006). At 88–91 amino acids, members of the SAUR19–24 clade are among the smallest SAUR proteins. In general, larger SAURs have N- or C-terminal extensions, with their middle regions being most closely related to SAUR19–24 (Figure S1). As such, SAUR19–24 may represent a ‘minimal’ or ‘core’ SAUR sequence

Feature

The SAUR19–24 genes of Arabidopsis are found in a tandem array on chromosome V and encode highly related (93–96% identity) proteins. Prior phylogenetic analysis revealed that SAUR19–24 form a unique clade in Arabidopsis (Jain et al., 2006). At 88–91 amino acids, members of the SAUR19–24 clade are among the smallest SAUR proteins. In general, larger SAURs have N- or C-terminal extensions, with their middle regions being most closely related to SAUR19–24 (Figure S1). As such, SAUR19–24 may represent a ‘minimal’ or ‘core’ SAUR sequence

Feature

The SAUR19–24 genes of Arabidopsis are found in a tandem array on chromosome V and encode highly related (93–96% identity) proteins. Prior phylogenetic analysis revealed that SAUR19–24 form a unique clade in Arabidopsis (Jain et al., 2006). At 88–91 amino acids, members of the SAUR19–24 clade are among the smallest SAUR proteins. In general, larger SAURs have N- or C-terminal extensions, with their middle regions being most closely related to SAUR19–24 (Figure S1). As such, SAUR19–24 may represent a ‘minimal’ or ‘core’ SAUR sequence

Feature

The SAUR19–24 genes of Arabidopsis are found in a tandem array on chromosome V and encode highly related (93–96% identity) proteins. Prior phylogenetic analysis revealed that SAUR19–24 form a unique clade in Arabidopsis (Jain et al., 2006). At 88–91 amino acids, members of the SAUR19–24 clade are among the smallest SAUR proteins. In general, larger SAURs have N- or C-terminal extensions, with their middle regions being most closely related to SAUR19–24 (Figure S1). As such, SAUR19–24 may represent a ‘minimal’ or ‘core’ SAUR sequence

Feature

The SAUR19–24 genes of Arabidopsis are found in a tandem array on chromosome V and encode highly related (93–96% identity) proteins. Prior phylogenetic analysis revealed that SAUR19–24 form a unique clade in Arabidopsis (Jain et al., 2006). At 88–91 amino acids, members of the SAUR19–24 clade are among the smallest SAUR proteins. In general, larger SAURs have N- or C-terminal extensions, with their middle regions being most closely related to SAUR19–24 (Figure S1). As such, SAUR19–24 may represent a ‘minimal’ or ‘core’ SAUR sequence

Feature

The bHLH140 protein is characterized by the presence of the QAR motif, which contains the amino acids glutamine (Q), alanine (A) and arginine (R) at positions 5, 9 and 13, respectively, within the bHLH domain (Heim et al., 2003). A QAR motif search in the Arabidopsis genome revealed a group of 14 bHLH140-related genes that can be divided into five sub-groups based on sequence similarities. Phylogenetic analysis grouped bHLH140, LAX1 and ba1 into a separate sub-group of the QAR motif bHLH genes

Feature

LAX1 and BA1, two highly conserved bHLH transcription factors, are important regulators of inflorescence and shoot branching in rice and maize, respectively (Komatsu et al., 2003; Gallavotti et al., 2004). We performed a BLAST search using the bHLH domain amino acid sequence to identify the genes most closely related to LAX1 in Arabidopsis thaliana, and found that bHLH140 showed the highest sequence similarity to both LAX1 and BA1, with 81% amino acid identity within the bHLH domain

Feature

As LAX1 and ba1 are intronless and have open reading frames (ORFs) of 648 and 660 bp, respectively, the bHLH140 gene was expected to contain a short ORF of similar length. However, the gene model for At5g01310 in the TAIR database (http://www.arabidopsis.org/) comprises six exons and five introns encompassing a coding region of 2739 bp. To investigate this discrepancy, we performed 3′ RACE PCR analysis using a forward primer specific for the first exon of At5g01310.1. All eight clones tested contained a poly(A) tail of 17–28 bp in length starting in the region 100–202 bp after the end of the annotated first exon (Figure S1). These data demonstrated that bHLH140 is an intronless gene harboring an ORF 516 bp in length (Figure S2), corroborating recent results by Woods et al. (2011

Feature

A de novo motif search using Amadeus (Linhart et al., 2008) of the 300 bp region upstream of the annotated transcription start site of the putative LEC1 target genes revealed two over-represented octameric motifs: a G box-containing motif that was present in 54% and a CCAAT-box motif that was present in 47% of all putative LEC1 target genes (Table 1). The calculated sequence logos are given in Figure 6. Additional simple motif search analysis revealed the presence of a CCAAT pentamer in 58% of the putative LEC1-bound promoters (−300 bp) using promoter sequences from TAIR9 (http://www.arabidopsis.org).

Feature

PIF5 has been shown previously to bind directly to the G-box DNA motif (5′-CACGTG-3′) (Hornitschek et al., 2009). We therefore analyzed PIF5 binding peaks, defined as 200 bp centered to the peak summit, for the presence of this sequence and of the E-box (5′-CANNTG-3′), a degenerated G-box that is also bound by bHLH transcription factors. Almost all PIF5 peaks contained an E-box (96%), the majority of which was a G-box (55%) (Figure 1c). Using motif-based sequence analysis tools (http://meme.sdsc.edu/meme/intro.html) we confirmed that the G-box is highly over-represented in PIF5 peaks. G-boxes were enriched in the center of PIF5 peaks, a finding that suggested that they mediate DNA binding

Feature

An earlier analysis of 25 sequences of U2AF35 proteins in plants and animals identified a domain in the C-terminal end of the plant proteins that was not present in the animal proteins (Wang and Brendel, 2006). blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches using plant U2AF35 sequences also identified this domain in non-photosynthetic protozoans such as Plasmodium and Toxoplasma; however, in most cases the domain was less conserved in the protozoans than in plants. Further analysis of U2AF35 sequences from flowering plants, gymnosperms, moss, algae and animals (84 sequences in total) confirmed the presence of this domain in photosynthetic eukaryotes but not in animals (Figure S2). These 84 U2AF35 homologs form several sub-families in a maximum likelihood gene tree (Figure S3). Although the animal sequences lack the conserved C-terminal PSD, they form a sister group with high bootstrap support values with algal and moss U2AF35 sequences

Feature

An earlier analysis of 25 sequences of U2AF35 proteins in plants and animals identified a domain in the C-terminal end of the plant proteins that was not present in the animal proteins (Wang and Brendel, 2006). blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches using plant U2AF35 sequences also identified this domain in non-photosynthetic protozoans such as Plasmodium and Toxoplasma; however, in most cases the domain was less conserved in the protozoans than in plants. Further analysis of U2AF35 sequences from flowering plants, gymnosperms, moss, algae and animals (84 sequences in total) confirmed the presence of this domain in photosynthetic eukaryotes but not in animals (Figure S2). These 84 U2AF35 homologs form several sub-families in a maximum likelihood gene tree (Figure S3). Although the animal sequences lack the conserved C-terminal PSD, they form a sister group with high bootstrap support values with algal and moss U2AF35 sequences

Feature

To address this situation, we prepared labelled tenth-intron RNA and performed an electrophoretic mobility shift assay (EMSA) to test the binding of purified SR45 and U2AF35b recombinant proteins to intron 10 RNA. Both proteins bound to intron 10 and the binding increased with increasing concentration of protein (Figure 6b, P1). To determine which part of the intron binds to these two proteins, we divided the entire intron into three parts (P2, P3, and P4) (Figure 6a). P2 contains the first 308 nucleotides, P3 contains the middle part of the intron, nucleotides 309–604, with a 3′ splice site (AG) at the end, and P4 consists of the remaining 338 nucleotides of SR30 intron 10 with a 3′ splice site (AG) at the end (Figure 6a). Five fmoles of labelled RNA from each part were incubated with increasing concentrations of purified SR45 and U2AF35b proteins and the binding was analyzed by EMSA. As shown in Figure 6(b) (P2, P3 and P4), the 5′ region of the intron (P2) binds to SR45 and RNA from P3 and P4 did not bind. In contrast, U2AF35b bound to P3 and P4 RNA but no binding was observed with P2. We then addressed the specificity of the P2 RNA binding to SR45 and U2AF35b binding to P3 and P4 RNA by adding an excess of corresponding cold RNA to the binding assay. As shown in Figure 6(c), complex formation was observed between the SR45 protein and P2 RNA (Lane 2), whereas addition of cold competitor RNA completely eliminated the binding (Lane 3). Similarly, when P3 and P4 RNAs were used with U2AF35b, RNA–protein complex formation was observed (Lane 5 and Lane 8), and addition of cold competitor RNA completely abolished the binding (Lanes 6 and 9). These results indicate that the interaction between SR45 and P2 RNA and U2AF35b with P3 and P4 RNAs is specific. Furthermore, our data clearly demonstrate that SR30 intron 10 binds to both SR45 and U2AF35b with SR45 binding to the 5′ region of the intron (P2) and U2AF35b binding to the other two regions (P3 and P4), each containing an experimentally verified 3′ splice site

Feature

To address this situation, we prepared labelled tenth-intron RNA and performed an electrophoretic mobility shift assay (EMSA) to test the binding of purified SR45 and U2AF35b recombinant proteins to intron 10 RNA. Both proteins bound to intron 10 and the binding increased with increasing concentration of protein (Figure 6b, P1). To determine which part of the intron binds to these two proteins, we divided the entire intron into three parts (P2, P3, and P4) (Figure 6a). P2 contains the first 308 nucleotides, P3 contains the middle part of the intron, nucleotides 309–604, with a 3′ splice site (AG) at the end, and P4 consists of the remaining 338 nucleotides of SR30 intron 10 with a 3′ splice site (AG) at the end (Figure 6a). Five fmoles of labelled RNA from each part were incubated with increasing concentrations of purified SR45 and U2AF35b proteins and the binding was analyzed by EMSA. As shown in Figure 6(b) (P2, P3 and P4), the 5′ region of the intron (P2) binds to SR45 and RNA from P3 and P4 did not bind. In contrast, U2AF35b bound to P3 and P4 RNA but no binding was observed with P2. We then addressed the specificity of the P2 RNA binding to SR45 and U2AF35b binding to P3 and P4 RNA by adding an excess of corresponding cold RNA to the binding assay. As shown in Figure 6(c), complex formation was observed between the SR45 protein and P2 RNA (Lane 2), whereas addition of cold competitor RNA completely eliminated the binding (Lane 3). Similarly, when P3 and P4 RNAs were used with U2AF35b, RNA–protein complex formation was observed (Lane 5 and Lane 8), and addition of cold competitor RNA completely abolished the binding (Lanes 6 and 9). These results indicate that the interaction between SR45 and P2 RNA and U2AF35b with P3 and P4 RNAs is specific. Furthermore, our data clearly demonstrate that SR30 intron 10 binds to both SR45 and U2AF35b with SR45 binding to the 5′ region of the intron (P2) and U2AF35b binding to the other two regions (P3 and P4), each containing an experimentally verified 3′ splice site

Feature

To address this situation, we prepared labelled tenth-intron RNA and performed an electrophoretic mobility shift assay (EMSA) to test the binding of purified SR45 and U2AF35b recombinant proteins to intron 10 RNA. Both proteins bound to intron 10 and the binding increased with increasing concentration of protein (Figure 6b, P1). To determine which part of the intron binds to these two proteins, we divided the entire intron into three parts (P2, P3, and P4) (Figure 6a). P2 contains the first 308 nucleotides, P3 contains the middle part of the intron, nucleotides 309–604, with a 3′ splice site (AG) at the end, and P4 consists of the remaining 338 nucleotides of SR30 intron 10 with a 3′ splice site (AG) at the end (Figure 6a). Five fmoles of labelled RNA from each part were incubated with increasing concentrations of purified SR45 and U2AF35b proteins and the binding was analyzed by EMSA. As shown in Figure 6(b) (P2, P3 and P4), the 5′ region of the intron (P2) binds to SR45 and RNA from P3 and P4 did not bind. In contrast, U2AF35b bound to P3 and P4 RNA but no binding was observed with P2. We then addressed the specificity of the P2 RNA binding to SR45 and U2AF35b binding to P3 and P4 RNA by adding an excess of corresponding cold RNA to the binding assay. As shown in Figure 6(c), complex formation was observed between the SR45 protein and P2 RNA (Lane 2), whereas addition of cold competitor RNA completely eliminated the binding (Lane 3). Similarly, when P3 and P4 RNAs were used with U2AF35b, RNA–protein complex formation was observed (Lane 5 and Lane 8), and addition of cold competitor RNA completely abolished the binding (Lanes 6 and 9). These results indicate that the interaction between SR45 and P2 RNA and U2AF35b with P3 and P4 RNAs is specific. Furthermore, our data clearly demonstrate that SR30 intron 10 binds to both SR45 and U2AF35b with SR45 binding to the 5′ region of the intron (P2) and U2AF35b binding to the other two regions (P3 and P4), each containing an experimentally verified 3′ splice site

Feature

The domains identified in this alignment were used to determine which part(s) of SR45 interact with the U2AF35 proteins. A series of deletion mutants of SR45 in a BiFC vector as fusions to YFPC was used in BiFC assays with the U2AF35/YFPN constructs. The SR45/YFPC constructs included SR45RS1, SR45RRM, SR45RS2, SR45RS1 + RRM and SR45RRM+RS2 (Figure 3a) (Ali et al., 2008). The SR45/YFPC constructs were tested with the YFPN constructs of U2AF35a, U2AF35b and U2AF35Ctrb. The protoplasts transfected with either the SR45RS1/YFPC or SR45RS2/YFPC construct and each U2AF35/YFPN construct showed fluorescence, which indicated an in vivo association of the proteins (Figure 3b–d, rows 1 and 3). However, protoplasts transfected with SR45RRM/YFPC and each U2AF35/YFPN construct did not show fluorescence (row 2). Furthermore, while the protoplasts transfected with the SR45RS2 + RRM/YFPC and each U2AF35/YFPN construct showed fluorescence (row 5), the protoplasts transfected with the SR45RS1 + RRM/YFPN construct showed fluorescence only when co-transfected with U2AF35Ctrb/YFPN (row 4). The fluorescence in protoplasts transfected with the SR45RS2/YFPC and U2AF35a/YFPN constructs (Figure 3b, row 2) appeared in smaller diffuse speckles throughout the nucleus as compared with the full-length SR45/YFPC construct with the U2AF35a/YFPN construct (Fig. 2, upper panels). While in protoplasts transfected with the SR45RS1/YFPC and U2AF35b/YFPN constructs, the fluorescence was more diffuse throughout the nucleus and speckles were very fine as compared with protoplasts transfected with the full-length SR45/YFPC or SR45RS2/YFPC (Compare Fig. 2 middle panels to Fig. 3c). The fluorescence in the protoplasts transfected with the SR45/YFPC sub-domain constructs and the U2AF35Ctrb appeared in most cases to be in larger speckles than when full-length SR45/YFPC was co-transfected with U2AF35Ctrb (compare Figure 2 lower panels with Figure 2d). These results suggest that although U2AF35 can interact with the SR45RS1 and SR45RS2 domains independently, other domains of the protein modulate the strength and specificity of this interaction. Furthermore, observed fluorescence with the SR45RS1 + RRM and U2AF35Ctrb constructs but not with the U2AF35a and U2AF35b constructs that contained the PSD, indicates that the C-terminal domain is likely to inhibit or interfere with their interaction with the SR45RS1 + RRM. In the case of the SR45RS1 + RRM construct, the RS1 domain is followed by the RRM while in the case of SR45RS2 + RRM construct, the RRM precedes the RS2 domain. It appears that when the C-terminal PSD is present, as in U2AF35a and full-length U2AF35b, U2AF35 cannot bind to the SR445RS1 + RRM. Whereas U2AF35 without this domain (U2AF35Ctrb) can bind, which leads to the possibility that there is steric hindrance between the SR45RRM domain and the U2AF35 C-terminal portion that does not allow association with the SR45RS1 domain but does allow interaction with the SR45RS2 domain. Possibly, U2AF35 binds to the SR45RS2 domain in the full-length protein leaving the SR45RS1 domain to interact with other proteins

Feature

SR45 has a modular structure comprised of an N-terminal RS domain (RS1), a central RRM domain and a C-terminal RS domain (RS2). We blasted the Phytozome databases with Arabidopsis SR45 and recovered 24 SR45 homologs (including AtSR45). Figure S4 shows an alignment of the homologs and the location of the domains. These protein sequences formed three major groupings: dicots, monocots and mosses (Figure S5).

Feature

To identify a cis-regulatory motif required for regulation by REV, we used MEME (meme.sdsc.edu) and compared the top 50 immunoprecipitated regions from both ChIP-Seq experiments. This analysis yielded the sequence motif AT[G/C]AT (Figure 1d). The AT[G/C]AT sequence represents the inner core of the inverted palindromic sequence GTAAT[G/C]ATTAC, which was identified as in vitro binding sequence for HD-ZIPIII proteins (Sessa et al., 1998). Of the 286 high confidence peaks, identified in both ChIP-Seq experiments, we find about 60% to be located in the 5′ promoter region of putative target genes and about 30% in the 3′ region (Figure 1e). Binding in the coding sequence or in the untranslated regions (UTRs) was seldom detected

Feature

DPA4 is a member of the B3 superfamily and a RAV [Related to ABSCISIC ACID-INSENSITIVE 3 (ABI3)/VIVIPAROUS1 (VP1)] transcriptional repressor containing the repressive motif described for B3 transcription factors (L/VRLFGV N/D M/L/V) in the variety VRLFGVNL

Feature

Sequences close to the GCCCR putative TCP20 binding site (Kosugi and Ohashi, 2002; Li et al., 2005) were found upstream of the LOX2 transcriptional start site at -1076 (GCCCG) and at -2799 bp (TGGGCC) (Fig. 4A).

Feature

The At4g11920 gene consists of nine exons and eight introns (Figure 4B) and encodes a putative CDH1/CCS52A2/FZR1 protein (CDH1, also known as HCT1 for Homolog of CDC Twenty; CCS52A2, also known as a 52 kDa protein encoded by a Cell Cycle Switch gene), which contains WD40 repeats and is a component of APC/C, acting a co-activator and substrate recognizer

Feature

At4g23940 was annotated in TAIR (http://arabidopsis.org/) as a member of the FtsH family, and contains conserved Walker A and Walker B motifs and a SRH. arc1 harbors a mutation in the first residue of the Walker B motif (S524F) (Figure 1b and Figure S2). The C-terminal region contains a domain corresponding to the proteolytic domain of FtsH, but lacks the histidine residues required for Zn-binding and protease activity (Zhang et al., 2010) (Figure 1b, asterisk). This observation was noted previously in a comparative genomic analysis by Sokolenko et al. (2002), who consequently designated At4g23940 as FtsHi1 (i for protease-inactive). In accordance with their nomenclature, we henceforth refer to At4g23940 as FtsHi1, and the arc1 allele as ftsHi1-1

Feature

we identified the SWI2/SNF2 subgroup chromatin remodeling ATPases from 21 plant species. This revealed 29 homologs for the MINU family, which includes Arabidopsis MINU1 (CHR12; At3g06010) ... An average number of 1.3 MINU genes were present per plant species. A similar low level of gene duplication was observed for the other two SWI2/SNF2 ATPases (Figure S1). Phylogenetic analyses confirmed that the plant SWI2/SNF2 subgroup ATPases (Flaus et al., 2006) fall into three separate clades: SYD, BRM and MINU (Figures 1a and S1

Feature

we identified the SWI2/SNF2 subgroup chromatin remodeling ATPases from 21 plant species. This revealed 29 homologs for the MINU family, which includes Arabidopsis ... MINU2 (CHR23; At5g19310) ... An average number of 1.3 MINU genes were present per plant species. A similar low level of gene duplication was observed for the other two SWI2/SNF2 ATPases (Figure S1). Phylogenetic analyses confirmed that the plant SWI2/SNF2 subgroup ATPases (Flaus et al., 2006) fall into three separate clades: SYD, BRM and MINU

Feature

A comprehensive mass spectrometry analysis of recombinant – E. coli-synthesised – CPK proteins revealed in vitro phosphorylation of CPK28 at serines S228, S318 within the protein kinase domain and S495 in the C-terminal calmodulin-like domain (Figure S5a) (Hegeman et al., 2006). To assess potential in planta phosphorylation of CPK28 at these sites, Strep-tagged CPK28 and its inactive variant CPK28-D188A were transiently expressed in Arabidopsis leaf mesophyll protoplasts, derived from cpk28-1 to exclude potential phosphorylation by endogenous CPK28. After affinity-purification, phospho-peptides were analysed by mass spectrometry. Phosphorylated CPK28 peptides encompassing S318 (LTAAQALpSHAWVR) and S495 (IpSLHEFR) accumulated about 25-fold (S318) and 110-fold (S495), respectively, in the presence of active CPK28 compared with inactive CPK28-D188A, indicating in vivo auto-phosphorylation of these sites (Figure S5). Phosphorylated peptide encompassing S228 (FHDIVGpSAYYVAEPVLK) increased about four-fold upon expression of the active kinase ... To assess the functional relevance of these phosphorylation sites, we generated CPK28 kinase variants with single amino acid substitutions S to D (putative phospho-mimic aspartate) or S to A (unphosphorylatable alanine) via site directed mutagenesis. In vitro kinase activity was evaluated as described above (Figure S6). The amino acid substitutions at S228 and S318 resulted in a 70–80% reduced, but still calcium-dependent activity, compared to the wild-type enzyme, irrespective of the mutation introduced. In contrast, amino acid substitution at site S495 did not alter the in vitro protein kinase activity of CPK28

Feature

RPX promoter ... At a 90% confidence cut-off level, we identified a region from -141 to -127 bp (GGGTGTTGACGTGTC) upstream of the transcription start site that was annotated as a MYC_MYB binding site, covering multiple elements including potential bZIP and MYB recognition sites

Feature

To determine the cis-elements that direct vascular expression of RPX, we performed a promoter deletion study. Shortening the RPX promoter to 385 bp upstream of the start codon (which included 163 bp of the promoter and the 222 bp 5' UTR) did not affect vascular expression

Feature

To identify the DNA-binding site of RPX, we performed a motif-based sequence analysis of the upstream regions of the 55 proteasome genes. This analysis identified a novel cis-element, (T/A)(A/T/G)(A/T/C)TGGGC(C/G)(T/G/A)N, which we named proteasome-related cis-element (PRCE) (Figure 5a). We found that the PRCE motif is mainly located within the first 200 bp upstream of the transcription start site. To test whether the PRCE element is also present in other species, we analyzed 1000 bp promoter regions of proteasome subunit-encoding genes ... RPX binds the PRCE element

Feature

RPX promoter ... Four novel deletion constructs were generated to test the importance of the identified region for RPX expression. Partial or complete deletion of the 5' UTR (Figure 2a) did not affect vascular expression. However, removing the MYC_MYB region from the 163 bp promoter by shortening it to 119 bp completely abolished GUS staining

Feature

To gain mechanistic insight into the function of miR408, we studied its regulation in the seedling stage. Searching the proximal promoter regions (from -1 to -600 upstream the TSS) revealed an array of 10 putative GTAC motifs. The tetranucleotide GTAC was found to be the core sequence of copper-response elements in Chlamydomonas, Arabidopsis and Barbula unguiculata

Feature

To identify the regions of VQ9 responsible for their interaction, we also performed directed yeast two-hybrid analyses. As shown in Figure S2b, deletion of the N terminus (amino acids 1–150, including the VQ motif) of VQ9 completely abolished the WRKY8–VQ9 interaction. To clarify whether the short VQ motif was required for the interaction, we generated a mutant VQ9 (VQ9ΔVQ motif) in which the conserved VVQK residues in the VQ motif were replaced by EDLE. The yeast two-hybrid assay showed that there was no interaction in yeast cells harboring both the mutant VQ9ΔVQ motif prey and WRKY8 bait vectors (Figure S2b), suggesting that the VQ motif of VQ9 is critical for the WRKY8–VQ9 interaction

Feature

To further specify the regions of WRKY8 required for the interaction with VQ9, several truncated WRKY8 variants were fused to the Gal4 DNA-binding domain. As shown in Figure S2a, the middle region of WRKY8 (145 amino acids, from position 100 to 244, spanning the WRKY domain and zinc-finger motif) was essential for the interaction with VQ9, as the truncated WRKY8 variants with further deletions of amino acids from position 100 to 188 or with a site-mutated WRKY domain or zinc-finger motif failed to interact with VQ9

Feature

To test whether serine residues corresponding to BSK1 Ser230 are phosphorylated by BRI1 in other BSKs (Figure 6c), we mutated BSK6 Ser210 and BSK8 Ser213 to alanine and monitored in vitro phosphorylation of the mutant proteins by BRI1. In contrast to phosphorylation of GST–BSK1S230A, phosphorylation of GST–BSK6S210A and GST–BSK8S213A by MBP–BRI1-KD was not significantly reduced compared to the wild-type proteins (Figure 6b), indicating that the site preferentially phosphorylated by BRI1 varies among BSKs and suggesting the existence of various mechanisms of BSK regulation by BRI1

Feature

To test whether serine residues corresponding to BSK1 Ser230 are phosphorylated by BRI1 in other BSKs (Figure 6c), we mutated BSK6 Ser210 and BSK8 Ser213 to alanine and monitored in vitro phosphorylation of the mutant proteins by BRI1. In contrast to phosphorylation of GST–BSK1S230A, phosphorylation of GST–BSK6S210A and GST–BSK8S213A by MBP–BRI1-KD was not significantly reduced compared to the wild-type proteins (Figure 6b), indicating that the site preferentially phosphorylated by BRI1 varies among BSKs and suggesting the existence of various mechanisms of BSK regulation by BRI1

Feature

MAIN may be targeted to the nucleus by a predicted nuclear localization sequence (NLS) containing the amino acid sequence KRKRR (Brameier et al., 2007; Tsugeki et al., 2009), a fusion construct was generated in which a stop codon was introduced before this sequence. This fusion was no longer targeted to the nucleus, but remained in the cytoplasm (Figure S3). After modifying the NLS from KRKRR to KLNQR, the fusion protein also localized to the cytoplasm (Figure 2b), indicating that the KRKRR sequence is a functional NLS

Feature

An in silico analysis using Scan Prosite (de Castro et al., 2006) allowed detection of potential SnRK1 consensus phosphorylation sites in ... AtKRP6 ... that displayed a common putative phosphorylation site in the CDK/cyclin interaction domain (Thr152/Thr151 at the N–terminal end of the domain). Interestingly, AtKRP6 also contained a second SnRK1 consensus phosphorylation site at Ser91

Feature

A tandem mass spectrometry (MS/MS) analysis of in vitro phosphorylated AtKRP6 ... indicated that peptides containing the common consensus SnRK1 target site previously identified in silico (VRKTPT152AAEI in AtKRP6 ... were phosphorylated ... however, doubt remained regarding AtKRP6 as the peptide contained two closely located threonine residues (Thr150 and Thr152). To resolve this ambiguity, a site-directed mutagenesis strategy was used and the following AtKRP6 mutated forms were produced: AtKRP6T150A, AtKRP6T150D, AtKRP6T152A and AtKRP6T152D (Figure S1). In the reconstituted medium, both the AtKRP6T150A and AtKRP6T150D mutant forms exhibited phosphorylation on Thr152, whereas AtKRP6T152A and AtKRP6T152D did not show Thr150 phosphorylation

Feature

An in silico analysis using Scan Prosite (de Castro et al., 2006) allowed detection of potential SnRK1 consensus phosphorylation sites in ... AtKRP7 ... that displayed a common putative phosphorylation site in the CDK/cyclin interaction domain (Thr152/Thr151 at the N–terminal end of the domain

Feature

Mutated yeast cdc28 cells, which are unable to divide at a restrictive temperature (Figure 4b), were complemented by AtCDKA;1–AF, leading to yeast cell multiplication (Figure 4c). The complemented yeast cells were then transformed with either WT or mutated AtKRP6. Interestingly, AtKRP6WT and AtKRP6T152A appeared to block cell division (Figure 4e,g), thus confirming the inhibitory function of AtKRP6 on cell division. In contrast, the phosphorylation-mimetic form AtKRP6T152D did not alter the complementation process (Figure 4i), strongly suggesting that the phosphorylation event abolishes the AtKRP6 inhibitory effects on AtCDKA;1

Feature

A mutant version of YFP-CPD45N1, which contains the S90F mutation present in cpd45, was incubated with both the wild type (lanes 5, 6, and 7 of Figure 6b) and the mutant ARC5 promoter (lane 10 of Figure 6b). No up-shift was observed, suggesting that this mutation abolishes the DNA-binding activity of YFP-CPD45N1 in vitro. Most homologs of FHY3/CPD45 in other species have an A residue at the position corresponding to S90 in FHY3/CPD45, which is structurally more similar to the S residue than the F residue (Figure S6). The S90 residue exists in a conserved FAR1 DNA-binding domain (Figure S2c) and many of the residues flanking S90 are highly conserved (Figure S6). Since the S to F mutation could cause a significant change in protein structure, it may severely affect the structure of FHY3/CPD45 and it is likely that the mutated FHY3/CPD45 also loses its DNA-binding activity in vivo

Feature

FRS4/CPD25 ... belong to the FRS family. A phylogenetic analysis using the protein sequence of the DNA-binding domain of members of this gene family indicated that FHY3/CPD45 was most closely related to FAR1, and that FRS4/CPD25 was also closely related to these two proteins

Feature

FHY3/CPD45 belong to the FRS family. A phylogenetic analysis using the protein sequence of the DNA-binding domain of members of this gene family indicated that FHY3/CPD45 was most closely related to FAR1

Feature

To further understand how FHY3/CPD45 activates the expression of ARC5, the FBS and FBL motifs in the ARC5 promoter were mutated individually or in pairs and analyzed by EMSA (Figure 7a). For the promoter regions with a single mutation, all the probes were shifted in a manner similar to that of the wild type, except that they migrated somewhat faster through the gel. The mobility of these three probes varied slightly. Probes bearing the m3 mutation ran fastest and those with the m1 mutation ran slowest. Three mutated probes each containing a pair of mutations (i.e. only one FBS or FBL motif was not mutated) were also shifted and ran even faster. A part of the probe with the m1m3 mutation pair or with the m2m3 mutation pair was not shifted. The three probes bearing pairs of mutations also ran at different speeds. The probe with the m2m3 mutation ran faster than the other two probes and contained more unshifted DNA. The probe with the m1m2 mutations ran slowest and had no unshifted DNA. The increased migration of these probes could be due to reduced binding of FHY3/CPD45 and earlier disassociation. These data indicate that FHY3/CPD45 can bind to all three motifs, but that the strength of binding varies, with binding to the third motif (-169 to -163) being strongest and to the first (-232 to -227) weakest

Feature

To further understand the role of the FBS motif and the two FBL motifs in FHY3/CPD45-mediated gene activation, the single and double mutant forms of the ARC5 promoter described above were fused to GUS and the effect of these mutations on the ability of the promoter to drive gene expression was tested (Figure 7b). Whereas the m1 and m2 mutations had almost no effect on the activity of the promoter, the m3 mutation resulted in reduced promoter activity. Furthermore, the m1m2 and m1m3 mutation pairs also reduced the activity of the promoter, and the m2m3 mutation abolished promoter activity. These data are in agreement with the EMSA results and suggest that the second and third FHY3/CPD45-binding sites are essential for the activity of the ARC5 promoter

Feature

we analyzed the promoter region of ARC5 and identified one FBS motif and two ‘ACGCGC’ FBS-like (FBL) motifs (Figures S5a and 6a) (Lin et al., 2007). The two FBL motifs (from -232 to -227 and -182 to -177 bp) are in the forward direction, whereas the single FBS motif (-169 to -163 bp) is in the reverse

Feature

To confirm that the degradation of AtMBP-1 is mediated by a ubiquitin-dependent mechanism, proteins were extracted from 8-day-old 35S::AtMPB-1-YFP transgenic seedlings and WT seedlings in the presence of MG132, and ubiquitinated AtMBP-1 was detected on immuno-blots using GFP- and ubiquitin-specific antibodies Figure 5(c). In these assays, GFP antibodies detected high molecular mass proteins that migrated more slowly than AtMBP-1 and cross-reacted with anti-Ub, a finding that indicated that AtMBP-1 is ubiquitinated in vivo

Feature

The LOS2 transcript includes a second AUG translation initiation codon at amino acid position +93 relative to the first in-frame AUG ... Crude protein samples extracted from transgenic plants that express these gene constructs were analyzed by immunoblot assays probed with anti-GFP. As shown in Figure 2(b), two protein bands, with apparent molecular weights of approximately 78 kDa and 67 kDa, were detected in extracts from plants that express the 35S::LOS2-YFP construct. These molecular masses correspond to the predicted sizes of LOS2–YFP and AtMBP-1–YFP fusion proteins, respectively ... This shows that expression of LOS2 mRNA can be differentially translated to produce two protein isoforms in vivo that correspond with full-length LOS2 and truncated AtMBP-1

Feature

As different functions have been attributed to the radical SAM domain and the HAT domain of ELO3 and its homologs in other organisms, we further tested whether a single domain could complement the elo3-14 phenotype. No rescue of the elo3-14 mutant was observed when we over-expressed either domain alone under the CaMV35S promoter, suggesting that a complete ELO3 protein is required, although we cannot exclude the possibility that the truncated proteins were non-functional

Feature

We tested the importance of the L1 box in the ACR4 promoter by generating plants in which GFP expression was driven by a full-length wild-type promoter or by two versions of the ACR4 promoter in which the L1 box had been mutated (Figure S4). As previously reported, the wild-type promoter drove GFP expression in the embryonic epidermis and root pole from the globular stage of development onwards (Gifford et al., 2003). In contrast, although both mutated versions of the promoter were able to drive expression of GFP in the embryonic root pole, neither resulted in a strong epidermal GFP signal in the embryo, suggesting that the L1 box in the ACR4 promoter is required for normal epidermal expression of ACR4 during embryogenesis

Feature

One Illumina control library and two ChIP-Seq libraries for 35S:FLAG-GR-KAN1 were sequenced. After filtering for read quality, sequencing reads were mapped to the Arabidopsis genome (TAIR10), resulting in the identification of 17402 peaks that were enriched in two independent ChIP-Seq experiments over the control sample. We subsequently limited our analysis to peaks showing at least three-fold enrichment. This dataset contains 4183 KAN1 bound regions. From a MEME-ChIP analysis (http://www.meme.sdsc.org) a VGAATAW motif was identified in 1802 of the 4183 regions (Figure 1B), corresponding to 3151 genes potentially regulated by KAN1 (see Dataset S1). These loci were equally distributed over the five Arabidopsis chromosomes, with a lack of enriched peaks in the centromeric regions (Figure 1C). A further analysis of the distribution of the peaks relative to the gene models revealed that the majority of binding sites were located within 1.0 kb upstream of the transcriptional start site (about 24%) or 1.0 kb downstream of the coding region (about 11%). Peaks were underrepresented in gene coding regions (Figure 1D).

Feature

While truncated BES1 with deletions of amino acids (aa) 89–140 still interacted with HAT1, BES1 deletion up to aa 198 reduced the interaction and deletion up to aa 272 largely abolished the interaction. The results suggested that aa 140–272 of BES1 are important for interaction with HAT1

Feature

we examined the promoter of DWF4 ... In the approximately 1800 bp DWF4 promoter region, there are four BRRE sites for BES1/BZR1 binding (Figure 6a). There is a putative HB-binding site (TAATAATTA) close to the -1780 bp BRRE site

Feature

To identify the domains in HAT1 that are required for the interaction, we examined the interactions between GST–BIN2 and a series of truncated HAT1 proteins. While deletions to amino acid 134 and 191 in HAT1 had no effect on the GST–BIN2 interaction, deletion to amino acid 233 of HAT1 abolished the interaction with BIN2 (Figure 3b and S3a). Taken together, a LZ motif in HAT1 mediates the interaction between HAT1 and BIN2

Feature

To test which domain of HAT1 is involved in the interaction with BES1, GST pull-down assays were performed with GST–BES1 and several truncated MBP–HAT1 (Figure 5b). When HAT1 was deleted up to aa 135, the interaction still existed. But HAT1 with deletion up to aa 192 did not interact with BES1. The region from aa 135 to aa 192 is the homeodomain (HD) in HAT1, which probably mediates the interaction between HAT1 and BES1

Feature

To examine whether yucasin directly inhibited conversion of IPyA to IAA by YUC, we performed in vitro Arabidopsis YUC1 enzyme assays using His-tagged AtYUC1 (YUC1-His) recombinant protein. Expression of YUC1-His in the soluble fraction of Escherichia coli extracts was confirmed using anti-AtYUC1 and anti-His6 antibodies (Figure S1). Recombinant YUC1-His enzyme with co-factors FAD and NADPH effectively converted IPyA to IAA (Figure 3a,b and Figure S2). High concentrations of IPyA had inhibitory effects on the reaction (Figure 3c), indicating feed-forward inhibitory regulation of YUC1-His enzyme(s) by substrate IPyA. In addition, non-enzymatic conversion of IPyA to IAA was observed in the absence of enzyme (Figure 3a) due to the extreme chemical instability of IPyA (Truelsen, 1973). Importantly, yucasin clearly inhibited YUC1-His activity in a dose-dependent manner (Figure 3b,d). Consistent with its structural similarity to methimazole, a substrate of FMO, these results suggest that yucasin is a substrate analog of FMO and functions as a competitive inhibitor of YUC1, with a higher binding affinity than the substrate IPyA

Feature

Lines expressing a Myc-tagged SOG1 under the control of its own promoter (PSOG1:SOG1-Myc) were either transferred to control medium or medium supplemented with H2O2. As described previously, immunoblotting using anti-Myc antibody detected two bands under control conditions (Figure 9A), with the upper band corresponding to SOG1 phosphorylated in a DNA stress–independent manner by a yet to be identified kinase (Yoshiyama et al., 2013). Upon H2O2 treatment, a third slowly migrating band appeared at a similar position as detected by zeocin treatment (Yoshiyama et al., 2013). This band disappeared when protein extracts were treated with the λ protein phosphatase (λPP), indicating that it corresponds to a phosphorylated form of SOG1

Feature

The pGBKT7 vectors containing a WRKY domain mutant or a zinc-finger domain mutant were used to analyze which domain of WRKY57 is responsible for interaction with the JAZ or IAA proteins. The results showed that the zinc-finger domain of WRKY57 was necessary for interaction with both the JAZ and IAA proteins

Feature

To investigate which region of IAA29 is required for interaction with WRKY57, we fused 10 truncated IAA29 variants to the AD domain of the pGADT7 vector. The interaction between these derivatives and the WRKY57 protein (as determined using a yeast two-hybrid assay) revealed that domain II was specifically responsible for the interaction

Feature

Of these 2040 peaks, more than half had a summit located between -200 and +200 bp from the translation start site with a maximum between -100 and 0 bp (Figure 3B), illustrating the molecular function of AN3 as a transcriptional coactivator in the regulation of gene expression. Additionally, a search for motifs using RSAT peak motifs (Thomas-Chollier et al., 2012) led to the identification of two significantly enriched motifs in the peak sequences: the tgaCACGTGgca motif containing the core G-box sequence (CACGTG) and the GAGA motif (GAGAGAGA) (Supplemental Figures 8B and 8C), a putative element of Arabidopsis core promoters

Feature

To test which domain of MED25 was responsible for the MED16 interaction, we generated four truncated forms of MED25 according to the domains for the yeast two-hybrid assay (Figure 3b). The results showed that both the MED25ΔQ-rich [1–681 amino acids (aa)] without a Q-rich domain and MED25ΔACID/Q-rich (1–558 aa) without ACID and a Q-rich domain interacted with YID1/MED16. This suggests that the ACID domain and the Q-rich domain are not used in this interaction (Figure 3b). However, both the MED25ΔvWF-A (N228–836 aa) without the vWF-A domain and MED25ΔvWF-A/ACID (N228–681 aa) without the vWF-A domain and ACID domain failed to interact with YID1/MED16, suggesting that the vWF-A domain is crucial for the interaction between YID1/MED16 and MED25

Feature

To clarify the contributions of individual bases of KBX to KAN1 binding, we performed EMSA with double-stranded oligonucleotides bearing point mutations throughout this sequence. Nucleotides at the first, third, fourth, and sixth positions were critical for high affinity binding in vitro (Figure 1). KAN1bd-GST bound equally well to the 6-bp consensus sequence GAATAA and to an 8-bp palindrome, GAATATTC, that appeared in 6 of the 50 selected sequences (Figure 1; Supplemental Figure 1). By contrast, the protein showed little affinity for the consensus binding site (AGATT) of the GARP protein ARR10

Feature

KAN1bd-GST was affinity purified and used for electrophoretic mobility shift assay (EMSA)–based PCR-assisted oligonucleotide selection. This experiment produced 50 nonredundant oligonucleotide sequences that contained one or more instances of the partly degenerate 6-bp motif GNATA(T/A), which we termed the KANADI box

Feature

The chloroplast localization of ANU10 was consistently predicted by several computational tools (see the Materials and methods), including TargetP (score=0.929) and Multiloc2 (score=0.57). ChloroP 1.1 predicted a chloroplast transit peptide in ANU10 and in most of its orthologues from other land plants

Feature

ANU10 and most of its orthologues were also predicted to have a transmembrane domain (Supplementary Fig. S4A, Supplementary Table S2), suggesting that these proteins are anchored to chloroplast membranes. As an example, SOSUI predicted a transmembrane domain spanning residues 421–443 in ANU10

Feature

To gain insight into the evolutionary origin of this protein family, HMMER searches were carried out using a profile made with the sequences of several ANU10 homologues from land plants. HMMER allowed the identification of some distantly related sequences in Cyanobacteria. In line with these results, a search for known domains in the Pfam database (Punta et al., 2012) yielded a low significance hit to a domain of unknown function (DUF4335) present in some cyanobacterial proteins. Together, these data indicate that ANU10 is conserved among land plants. However, unlike proteins such as CURT1A (Armbruster et al., 2013), which is functionally conserved in Cyanobacteria, the search for cyanobacterial orthologues of ANU10 did not yield obvious candidates

Feature

ANU10 is a single-copy gene in the nuclear genome of Arabidopsis. Because the gene is predicted to encode a protein of unknown function with no conserved domains, BLAST searches were carried out to identify similar protein sequences in public databases. Significant hits were found in the genomes of other higher plants and the moss Physcomitrella patens, but not in those of animals or other eukaryotes, including algae, suggesting that ANU10 belongs to a family of embryophyte-specific proteins

Feature

Pairwise comparisons of genes for searching best hits in genome datasets of different organisms can identify reciprocal best-hits and their clusters. Genes in such clusters are believed to be, at least in many cases, in orthologous relationships (Kristensen et al., 2011). Therefore, we explored a RID3-containing cluster of reciprocal best-hits by performing multiple pairwise comparisons between well-annotated genome databases of various organisms, and found that a RID3-containing cluster lay within the eukaryote domain (Figure 1A). We then used sequences in such a cluster for phylogenetic tree construction. As reference sequences of the phylogenic tree, we chose the Arabidopsis homolog most similar to RID3 and its best-hit sequences in some other genomes, in consideration of the usefulness of duplicated gene pairs to specify the root position in a phylogenic tree (Iwabe et al., 1989). As shown in Figure 1B, the sequences of a RID3-containing cluster formed one distinct clade, within which the topology largely agreed with the generally accepted evolutionary history of the eukaryote lineage, although the clade grouping the Caenorhabditis elegans and yeast sequences together seems to be influenced by “long-branch attraction,” a common artifact in sequence-based phylogenetic tree construction (reviewed by Bergsten, 2005). Nonetheless, the well-supported branch (100%) of the sequences in the cluster indicates that the member sequences, including RID3, IPI3 of budding yeast Saccharomyces cerevisiae (Saccharomyces), and Pro-1 of C. elegans, were orthologous to each other. This conclusion is consistent with the previous large-scale analysis for orthologous group identification (Tatusov et al., 2003; KOG0646). In addition, we found that the Arabidopsis closest homolog lay outside the clade of RID3 orthologs despite the much higher BLAST-based similarity (E-value: 8E-13) than that (4E-04) of IPI3 to RID3

Feature

It has been already pointed out that the N-terminal domain of GIF1 has homology with the SNH domain of the human co-activator SYT (Kim and Kende, 2004), which in turn mediates the interaction with human SWI/SNF ATPases

Feature

PSI1 has no homology to proteins of known function. A Blast search revealed that PSI1 is a member of a small protein family in Arabidopsis. PSI proteins exist in plants but not in metazoa or microbial organisms. Phylogenetic analysis revealed that the six Arabidopsis PSI proteins separate into two clades (Fig. 2a). We further characterized the three members of clade 1 that includes PSI1 (At1g34320), PSI2 (At1g30755) and PSI3 (At5g08660). PSI2 shares 40 % identity and PSI3 shares 50.4 % identity with PSI1

Feature

a regulator of G protein signaling, a transcription factor, a protein of unknown function (termed PSI1; At1g34320), a leucine-rich repeat receptor kinase with homology to SERK kinases, a MAP kinase and a protein with predicted ATPase activity

Feature

PSI3 ... PSI proteins are about 650 amino acids in length. An alignment of clade 1 PSI proteins from Arabidopsis, Ricinus communis (RcPSI1), Vitis vinifera (VvPSI1), Populus trichocarpa (PtPSI1), Oryza sativa (OsPSI1), Zea mays (ZmPSI1) and Physcomitrella patens (PpPSI1) revealed a high sequence conservation at the N-terminus (Supplemental Fig. S4) that conforms to a predicted myristoylation site except for the Physcomitrella patens homolog. This site is characterized by a conserved glycine at position 2 which is essential as attachment site for myristate. Except for PSI2 the PSI proteins possess a small amino acid at the 6th amino acid position and have a positively charged amino acid at position 7 characteristic for N-terminal myristoylation sites

Feature

PSI1 has no homology to proteins of known function. A Blast search revealed that PSI1 is a member of a small protein family in Arabidopsis. PSI proteins exist in plants but not in metazoa or microbial organisms. Phylogenetic analysis revealed that the six Arabidopsis PSI proteins separate into two clades (Fig. 2a). We further characterized the three members of clade 1 that includes PSI1 (At1g34320), PSI2 (At1g30755) and PSI3 (At5g08660). PSI2 shares 40 % identity and PSI3 shares 50.4 % identity with PSI1

Feature

PSI proteins are about 650 amino acids in length. An alignment of clade 1 PSI proteins from Arabidopsis, Ricinus communis (RcPSI1), Vitis vinifera (VvPSI1), Populus trichocarpa (PtPSI1), Oryza sativa (OsPSI1), Zea mays (ZmPSI1) and Physcomitrella patens (PpPSI1) revealed a high sequence conservation at the N-terminus (Supplemental Fig. S4) that conforms to a predicted myristoylation site except for the Physcomitrella patens homolog. This site is characterized by a conserved glycine at position 2 which is essential as attachment site for myristate. Except for PSI2 the PSI proteins possess a small amino acid at the 6th amino acid position and have a positively charged amino acid at position 7 characteristic for N-terminal myristoylation sites. Based on this conserved sequence PSI1 had been identified as a member of the Arabidopsis myristome

Feature

PSI1 has no homology to proteins of known function. A Blast search revealed that PSI1 is a member of a small protein family in Arabidopsis. PSI proteins exist in plants but not in metazoa or microbial organisms. Phylogenetic analysis revealed that the six Arabidopsis PSI proteins separate into two clades (Fig. 2a). We further characterized the three members of clade 1 that includes PSI1 (At1g34320), PSI2 (At1g30755) and PSI3 (At5g08660). PSI2 shares 40 % identity and PSI3 shares 50.4 % identity with PSI1

Feature

In order to test whether the predicted protein possesses MAP kinase activity, we produced a GST-fusion protein of the wild-type and two mutant versions of AtMPK10 in Escherichia coli and tested them with myelin basic protein (MBP) as substrates in in vitro kinase assays ( Figure 2A). The AtMPK10 AEF mutant was created by changing the conserved Threonine and Tyrosine residues of the TEY MAPK phosphorylation motif in the activation loop to Alanine (T218A) and to Phenylalanine (Y220F), respectively. The R89 mutant was created by changing one of the two conserved Lysine residues (88 and 89) of the ATP-binding loop to an Arginine (K89R). The wild-type AtMPK10 showed phosphorylation activity similar to the well-characterized group B MAPK AtMPK4, whereas no auto-phosphorylation activity and a strongly reduced substrate phosphorylation almost to background levels was detected with the AtMPK10 AEF version. In contrast, the exchange of only one of the two Lysine residues of the ATP-binding domain was not sufficient to abolish MPK10 kinase activity (R89). Thus, the predicted AtMPK10 exhibits kinase activity that depends on a functional phosphorylation motif and we considered the AEF version of MPK10 as a loss-of-function (LOF) version and used this in the further experiments

Feature

To identify genes with homology to HUA2 in the Arabidopsis genome, we performed BLAST searches with HUA2 nucleotide and protein sequences, and identified three genes that, together with HUA2, form the HUA2-LIKE (or HULK) gene family. The HULK family members share a conserved domain structure that includes a PWWP domain (Pfam: PF00855; named after the conserved Pro-Trp-Trp-Pro motif), putative nuclear localization signal (NLS) motifs (ELM: TRG_NLS_MonoCore_2), an RPR domain (SMART: SM000582; regulation of nuclear pre-mRNA) and a PRR domain (proline-rich region) of variable length (Figures 1a and S1). Among the HULKs, pairwise amino acid identities range from 50.7 to 86.3% and from 47 to 88.6% for the PWWP and RPR domains, respectively. HUA2 (AT5G23150) and HULK1 (AT5G08230) are distantly linked on chromosome 5, while HULK2 (AT2G48160) and HULK3 (AT3G63070) are adjacent to the distal telomeres of chromosomes 2 and 3, respectively. Indicative of a comparatively recent gene family expansion, HUA2/HULK1 and HULK2/HULK3 are present within segmental duplications arising from the most recent paleopolyploidy event in the Arabidopsis lineage (Blanc et al., 2003 ... A broader search for proteins with a similar domain organization to that in the HULK proteins retrieved sequences from plants within the sub-kingdom Embryophyta, but not in green algae, animals or fungi. Phylogenetic analyses using the PWWP and RPR domains of HULK homologs from evolutionary distant species revealed two well-supported clades represented by HUA2/HULK1 and by HULK2/HULK3 (Figure 1b, puzzle support of 0.98). The split is ancient, and probably occurred in the common ancestor of angiosperms. With the exception of Malus domestica, Brassica rapa and Populus trichocarpa, all species for which we identified more than one HULK homolog have members belonging to both the HUA2/HULK1 and HULK2/HULK3 clades (Figure S2). The presence of putative NLS motifs

Feature

To identify genes with homology to HUA2 in the Arabidopsis genome, we performed BLAST searches with HUA2 nucleotide and protein sequences, and identified three genes that, together with HUA2, form the HUA2-LIKE (or HULK) gene family. The HULK family members share a conserved domain structure that includes a PWWP domain (Pfam: PF00855; named after the conserved Pro-Trp-Trp-Pro motif), putative nuclear localization signal (NLS) motifs (ELM: TRG_NLS_MonoCore_2), an RPR domain (SMART: SM000582; regulation of nuclear pre-mRNA) and a PRR domain (proline-rich region) of variable length (Figures 1a and S1). Among the HULKs, pairwise amino acid identities range from 50.7 to 86.3% and from 47 to 88.6% for the PWWP and RPR domains, respectively. HUA2 (AT5G23150) and HULK1 (AT5G08230) are distantly linked on chromosome 5, while HULK2 (AT2G48160) and HULK3 (AT3G63070) are adjacent to the distal telomeres of chromosomes 2 and 3, respectively. Indicative of a comparatively recent gene family expansion, HUA2/HULK1 and HULK2/HULK3 are present within segmental duplications arising from the most recent paleopolyploidy event in the Arabidopsis lineage (Blanc et al., 2003 ... A broader search for proteins with a similar domain organization to that in the HULK proteins retrieved sequences from plants within the sub-kingdom Embryophyta, but not in green algae, animals or fungi. Phylogenetic analyses using the PWWP and RPR domains of HULK homologs from evolutionary distant species revealed two well-supported clades represented by HUA2/HULK1 and by HULK2/HULK3 (Figure 1b, puzzle support of 0.98). The split is ancient, and probably occurred in the common ancestor of angiosperms. With the exception of Malus domestica, Brassica rapa and Populus trichocarpa, all species for which we identified more than one HULK homolog have members belonging to both the HUA2/HULK1 and HULK2/HULK3 clades (Figure S2). The presence of putative NLS motifs

Feature

To identify genes with homology to HUA2 in the Arabidopsis genome, we performed BLAST searches with HUA2 nucleotide and protein sequences, and identified three genes that, together with HUA2, form the HUA2-LIKE (or HULK) gene family. The HULK family members share a conserved domain structure that includes a PWWP domain (Pfam: PF00855; named after the conserved Pro-Trp-Trp-Pro motif), putative nuclear localization signal (NLS) motifs (ELM: TRG_NLS_MonoCore_2), an RPR domain (SMART: SM000582; regulation of nuclear pre-mRNA) and a PRR domain (proline-rich region) of variable length (Figures 1a and S1). Among the HULKs, pairwise amino acid identities range from 50.7 to 86.3% and from 47 to 88.6% for the PWWP and RPR domains, respectively. HUA2 (AT5G23150) and HULK1 (AT5G08230) are distantly linked on chromosome 5, while HULK2 (AT2G48160) and HULK3 (AT3G63070) are adjacent to the distal telomeres of chromosomes 2 and 3, respectively. Indicative of a comparatively recent gene family expansion, HUA2/HULK1 and HULK2/HULK3 are present within segmental duplications arising from the most recent paleopolyploidy event in the Arabidopsis lineage (Blanc et al., 2003 ... A broader search for proteins with a similar domain organization to that in the HULK proteins retrieved sequences from plants within the sub-kingdom Embryophyta, but not in green algae, animals or fungi. Phylogenetic analyses using the PWWP and RPR domains of HULK homologs from evolutionary distant species revealed two well-supported clades represented by HUA2/HULK1 and by HULK2/HULK3 (Figure 1b, puzzle support of 0.98). The split is ancient, and probably occurred in the common ancestor of angiosperms. With the exception of Malus domestica, Brassica rapa and Populus trichocarpa, all species for which we identified more than one HULK homolog have members belonging to both the HUA2/HULK1 and HULK2/HULK3 clades (Figure S2). The presence of putative NLS motifs

Feature

To identify genes with homology to HUA2 in the Arabidopsis genome, we performed BLAST searches with HUA2 nucleotide and protein sequences, and identified three genes that, together with HUA2, form the HUA2-LIKE (or HULK) gene family. The HULK family members share a conserved domain structure that includes a PWWP domain (Pfam: PF00855; named after the conserved Pro-Trp-Trp-Pro motif), putative nuclear localization signal (NLS) motifs (ELM: TRG_NLS_MonoCore_2), an RPR domain (SMART: SM000582; regulation of nuclear pre-mRNA) and a PRR domain (proline-rich region) of variable length (Figures 1a and S1). Among the HULKs, pairwise amino acid identities range from 50.7 to 86.3% and from 47 to 88.6% for the PWWP and RPR domains, respectively. HUA2 (AT5G23150) and HULK1 (AT5G08230) are distantly linked on chromosome 5, while HULK2 (AT2G48160) and HULK3 (AT3G63070) are adjacent to the distal telomeres of chromosomes 2 and 3, respectively. Indicative of a comparatively recent gene family expansion, HUA2/HULK1 and HULK2/HULK3 are present within segmental duplications arising from the most recent paleopolyploidy event in the Arabidopsis lineage (Blanc et al., 2003 ... A broader search for proteins with a similar domain organization to that in the HULK proteins retrieved sequences from plants within the sub-kingdom Embryophyta, but not in green algae, animals or fungi. Phylogenetic analyses using the PWWP and RPR domains of HULK homologs from evolutionary distant species revealed two well-supported clades represented by HUA2/HULK1 and by HULK2/HULK3 (Figure 1b, puzzle support of 0.98). The split is ancient, and probably occurred in the common ancestor of angiosperms. With the exception of Malus domestica, Brassica rapa and Populus trichocarpa, all species for which we identified more than one HULK homolog have members belonging to both the HUA2/HULK1 and HULK2/HULK3 clades (Figure S2). The presence of putative NLS motifs

Feature

A proteomic analysis of Arabidopsis plasma membrane proteins identified a phosphorylated residue (Ser-177) close to the C terminus of the DUF1218-containing protein encoded by At4g31130 (Hem et al., 2007), suggesting that the N- and C-terminal regions plus the central loop of this protein are in the cytoplasm and the first and third loop are exposed to the lumenal/extracellular space (Fig. 1C). Several Cys residues (black boxes in Fig. 1A) in the transmembrane domains and in the luminal/extracellular loops are well conserved across the whole DUF1218 protein family. In addition, several polar amino acid residues are found along the transmembrane domains

Feature

ABS5 is annotated to encode a protein of 368 amino acids and protein sequence analysis revealed that ABS5 is likely a putative transcription factor belonging to the basic helix-loop-helix (bHLH) family [32]. In Arabidopsis, there are at least 147 members in the bHLH family and ABS5/T5L1 was previous annotated as bHLH30 [32

Feature

To test the functionality of the predicted NLS in atLSG1-2 ... we generated GFP fusions of the N- or C-terminal parts of ... atLSG1-2 and transformed them transiently in protoplasts ... the C-terminus of atLSG1-2 (atLSG1-2535–589) fused to GFP results in a GFP fluorescence merging with the mCherry signal of atFIB2 in the nucleus and nucleolus ... These results are consistent with the observation that the human orthologue also contains a signal for localization in the nucleus and nucleolus in its C-terminus (Reynaud et al., 2005). In addition, the N-terminal region of atLSG1-2 (amino acids 1–535) fused to GFP results in a dual localization of GFP in the nucleus and in the cytoplasm as well

Feature

To test the functional conservation of the A. thaliana LSG1-proteins, we complemented a yeast depletion strain for scLsg1 with proteins ectopically expressed under the MET25-promoter (Figure 1c). As expected, scLsg1 fully supports growth of the strain after addition of doxycycline, which starts the depletion by repressing endogenous scLsg1 transcription by binding to the tet-repressor. Cells harbouring only the empty vector show growth inhibition after 12 h of depletion because scLsg1 is essential (Figure 1c). The two A. thaliana LSG-proteins partially complement the depletion of scLsg1 as judged by a medium growth restoration between positive (scLsg1) and negative (empty vector) control (Figure 1c). The expression of atLSG1 in yeast was verified by reverse transcription polymerase chain reaction (RT-PCR) and western blot (Figure S2) using an antibody against a mixture of atLSG1-1 and atLSG1-2. The specificity of the polyclonal antibody was confirmed by western blot against total protein and the purified LSG-proteins (Figure S3). Thus, atLSG1-1 and atLSG1-2 can complement the depletion of scLsg1, suggesting that they might also be involved in ribosome biogenesis in plants

Feature

we generated GFP fusions of the N- or C-terminal parts of atLSG1-1 ... The fusion proteins with the N- or C-terminal parts of atLSG1-1 (amino acids 1–469 and 469–535, respectively) show a cytoplasmic distribution ... This result is not dependent on the position of GFP because both N- and C-terminal GFP fusions show the same localization (Figure 2b: atLSG1-2535–589–GFP and GFP– atLSG1-2535–589. GFP alone shows only a slight nuclear staining and an exclusion of the nucleolus

Feature

During protein purification of atLSG1-2 from E. coli we observed co-purification of 23S rRNA and after RNase treatment a specific fragment was protected by atLSG1-2, which was identified by sequencing to position 1650–1670. The homologous sequence was mapped onto the yeast ribosome (PDB 3U5H) and is in close proximity to the Nmd3 binding site (Figure 4a; Matsuo et al., 2014). We performed fluorescence anisotropy measurements using the homologous 21 nt 3′-fluorescein labelled RNA sequence from A. thaliana and the atLSG1 proteins (Figure 4b) to determine quantitative interaction values. We observed binding of both atLSG1-1 and atLSG1-2 to the RNA as the anisotropy increases. However, the KD for the atLSG1-2-RNA complex is 91 nm whereas the atLSG1-1–RNA interaction was around 20-fold weaker (2000 nm, Figure 4b). None of the proteins formed a complex with the recognition sequence of Emg1 within the rRNA (Wurm et al., 2010), which served as a negative control. These results show that both atLSG1 proteins bind to rRNA, but, however, atLSG1-2 has a higher affinity than atLSG1-1

Feature

We searched for orthologues of members of the YlqF/YawG family with a circular permuted order of GTPase motifs, namely Nog1, Nug1, Lsg1 and Nog2. Phylogenetic analysis of the identified sequences shows that the four proteins are clearly distinct and very ancient as previously proposed (Figure 1a; Ebersberger et al., 2014), because the according yeast protein is the base of each of the four clades. Several gene duplications are found for all four proteins, however, with the exception of Lsg1 found in Brachypodium distachyon and Oryza sativa, the multiple isoforms are the result of very recent gene duplications. This holds true for the two identified A. thaliana orthologues of scLsg1 (atLSG1-1, At2g27200; atLSG1-2, At1 g08410), which is known to function in the biogenesis of the large ribosomal subunit in yeast ... The two identified Lsg1 orthologues in A. thaliana are highly conserved especially in the functional GTPase domains (Figure S1), but do not contain the N-terminal positively charged region present in scLsg1 (Figure 1b). Further, the atLSG1-proteins differ in their C-terminal extension, which is highly charged in scLsg1 and atLSG1-2, but not in atLSG1-1 (Figure 1b). This extension contains negatively charged amino acids followed by a positively charged C-terminus (Figure 1b). Furthermore, using cNLS pred (Kosugi et al., 2009a,b), a weak nuclear localization signal (NLS) could be predicted for atLSG1-2 in its positively charged C-terminus (Figures 1b and S1

Feature

During protein purification of atLSG1-2 from E. coli we observed co-purification of 23S rRNA and after RNase treatment a specific fragment was protected by atLSG1-2, which was identified by sequencing to position 1650–1670. The homologous sequence was mapped onto the yeast ribosome (PDB 3U5H) and is in close proximity to the Nmd3 binding site (Figure 4a; Matsuo et al., 2014). We performed fluorescence anisotropy measurements using the homologous 21 nt 3′-fluorescein labelled RNA sequence from A. thaliana and the atLSG1 proteins (Figure 4b) to determine quantitative interaction values. We observed binding of both atLSG1-1 and atLSG1-2 to the RNA as the anisotropy increases. However, the KD for the atLSG1-2-RNA complex is 91 nm whereas the atLSG1-1–RNA interaction was around 20-fold weaker (2000 nm, Figure 4b). None of the proteins formed a complex with the recognition sequence of Emg1 within the rRNA (Wurm et al., 2010), which served as a negative control. These results show that both atLSG1 proteins bind to rRNA, but, however, atLSG1-2 has a higher affinity than atLSG1-1

Feature

To test the functional conservation of the A. thaliana LSG1-proteins, we complemented a yeast depletion strain for scLsg1 with proteins ectopically expressed under the MET25-promoter (Figure 1c). As expected, scLsg1 fully supports growth of the strain after addition of doxycycline, which starts the depletion by repressing endogenous scLsg1 transcription by binding to the tet-repressor. Cells harbouring only the empty vector show growth inhibition after 12 h of depletion because scLsg1 is essential (Figure 1c). The two A. thaliana LSG-proteins partially complement the depletion of scLsg1 as judged by a medium growth restoration between positive (scLsg1) and negative (empty vector) control (Figure 1c). The expression of atLSG1 in yeast was verified by reverse transcription polymerase chain reaction (RT-PCR) and western blot (Figure S2) using an antibody against a mixture of atLSG1-1 and atLSG1-2. The specificity of the polyclonal antibody was confirmed by western blot against total protein and the purified LSG-proteins (Figure S3). Thus, atLSG1-1 and atLSG1-2 can complement the depletion of scLsg1, suggesting that they might also be involved in ribosome biogenesis in plants

Feature

We searched for orthologues of members of the YlqF/YawG family with a circular permuted order of GTPase motifs, namely Nog1, Nug1, Lsg1 and Nog2. Phylogenetic analysis of the identified sequences shows that the four proteins are clearly distinct and very ancient as previously proposed (Figure 1a; Ebersberger et al., 2014), because the according yeast protein is the base of each of the four clades. Several gene duplications are found for all four proteins, however, with the exception of Lsg1 found in Brachypodium distachyon and Oryza sativa, the multiple isoforms are the result of very recent gene duplications. This holds true for the two identified A. thaliana orthologues of scLsg1 (atLSG1-1, At2g27200; atLSG1-2, At1 g08410), which is known to function in the biogenesis of the large ribosomal subunit in yeast ... The two identified Lsg1 orthologues in A. thaliana are highly conserved especially in the functional GTPase domains (Figure S1), but do not contain the N-terminal positively charged region present in scLsg1 (Figure 1b). Further, the atLSG1-proteins differ in their C-terminal extension, which is highly charged in scLsg1 and atLSG1-2, but not in atLSG1-1 (Figure 1b). This extension contains negatively charged amino acids followed by a positively charged C-terminus (Figure 1b). Furthermore, using cNLS pred (Kosugi et al., 2009a,b), a weak nuclear localization signal (NLS) could be predicted for atLSG1-2 in its positively charged C-terminus (Figures 1b and S1

Feature

We investigated MTERF9 conservation in different photosynthetic organisms by using the plaza database (http://bioinformatics.psb.ugent.be/plaza/), a comparative plant genomics resource. We identified 24 putative orthologues in green algae, mosses, lycopsids, monocotyledonous and dicotyledonous species (Fig. S3). target p1.1 predicted most of them to be chloroplastic, including the product of the rice orthologous gene (Os07g39430; cTP = 0.862; Fig. S3B). Besides, the ortholog protein from maize, ZmTERF9, was identified in the nucleoids of plastid leaves (Majeran et al. 2012). In comparison to MTERF9, the ZmTERF9 and the Os07g39430-encoded protein had a similar number of mTERF motifs (7) and residues (503 and 508, respectively), and their level of homology with MTERF9 (54.5 and 48.4% identity, respectively) was higher than that of MTERF9 with any A. thaliana paralog. In accordance with the plaza database, Arabidopsis lyrata AL8G20980 and Populus trichocarpa PT01G36520 proteins showed the highest levels of identity with MTERF9 (93.8 and 68.1%, respectively; Fig. S3A). Our phylogenetic analysis revealed that monocotyledonous, dicotyledonous and lower-plant MTERF9 orthologues were grouped into different clades (Fig. S3B). Apart from plants, MTERF9 displayed the highest homology with the members of the MTERF3 sub-family from metazoans (e.g. 24.4% identity and 56.5% similarity with human MTERF3; 21.1% identity and 54.7% similarity with Drosophila melanogaster mTERF3). A sequence-based alignment of A. thaliana MTERF9 and human MTERF3 proteins revealed the conservation of several hydrophobic residues within mTERF-motifs 1–4, described to form hydrophobic interactions required to stabilize the mTERF-motifs, as well as some conservation of proline residues (e.g. in mTERF-motifs 3, 4 and 6) involved in the right twist of the mTERF repeats (Spåhr et al. 2010; Fig. S4).

Feature

According to the GFP-fusion results from Babiychuk et al. (2011) and our own bioinformatic predictions using target p1.1 (http://www.cbs.dtu.dk/services/TargetP/), protein prowler (http://pprowler.itee.uq.edu.au/pprowler_webapp_1-2/) and ipsort (http://ipsort.hgc.jp/), MTERF9 is a chloroplast protein. In line with this, MTERF9 has been recently included as a component of the A. thaliana reference plastid proteome, according to Huang et al. (2013).

Feature

The altered responses to abiotic stress displayed by the mterf9 (see above) and mda1 mutants (Robles et al. 2012a) prompted us to analyze MDA1 and MTERF9 promoters in order to identify stress-related cis-elements. For this purpose, approximately 2-kb sequences located upstream of the transcription start sites of the MDA1, MTERF9, OTC and gamma-tubulin 2 (TUBG2) genes, the latter two used as controls, were analyzed using the PlantPAN server (http://plantpan.mbc.nctu.edu.tw/index.php). We identified all the putative binding sites for the plant transcription factors present in the promoters of MDA1 and MTERF9, whose predicted position and abundance are shown in Table S7, and a significant co-occurrence was detected for 59 of them. Next we sought among all the cis-elements found those previously described by its relationship with environmental stimulus responses in plants, and classified them into different categories: hormone (ABA, gibberellin acid, auxin or ethylene), abiotic-stress (drought, heat, low temperature, hypo-osmolarity or multiple stresses), sugar, light and anaerobic-responsive elements (Table S8). A similar number of different cis-elements was identified in MDA1 (29) and MTERF9 (32) promoters, which was higher than those found in the promoters of the OTC (21) and TUBG2 (17) (65 and 53% of those found in MTERF9, respectively; Table S8). Hence, unlike MDA1 or MTERF9, in the OTC and TUBG2 promoters we did not find low temperature-responsive elements, heat stress-responsive elements and hypo-osmolarity responsive elements, while the categories of sugar-responsive elements and ethylene-responsive elements where absent in OTC and TUBG2, respectively. Besides, the classes of multiple abiotic stresses-responsive elements and drought-responsive elements (DREs) and ABA-responsive elements showed a lower number of different cis-elements in OTC and TUBG2 than in MDA1 or MTERF9 (Table S8). The most representative classes in MDA1 and MTERF9 were those of the light-responsive elements (LRE; eight and six different sequences in MDA1 and MTERF9, respectively, and more than 30 LRE per promoter) and DRE (seven and five different sequences in MDA1 and MTERF9, respectively, and more than 20 sequences per promoter) (Tables S7 and S8). We found the significant co-occurrence of 21 putative abiotic-stress related elements in MDA1 and MTERF9 (Table S8). Hence, MDA1 and MTERF9 expression may be dependent on different environmental stimuli

Feature

It has been shown that proteins of the class II homeodomain leucine-zipper (HD-ZIPII) family from sunflower interact with DNA in a redox-sensitive manner (Tron et al., 2002). To test whether REV shows also redox-dependent DNA binding, we performed redox-sensitive DPI-ELISA experiments. Therefore, crude lysate of E. coli cells expressing HIS-tagged REV protein were prepared and incubated with streptavidin plates pre-loaded with biotinylated oligonucleotides containing the REV-binding site 1 of the WRKY53 promoter (W53-BS1). ELISA plates were then washed and subsequently incubated with HRP-tagged anti-HIS antibodies. Enhanced signal was detected in the control binding reaction (HIS-REV lysate versus a lysate from BL21 cells expressing the empty vector control), indicating that HIS-REV binds to the W53-BS1 element (Fig. 4A). As observed for the sunflower HD-ZIPII proteins (Tron et al., 2002), REV also showed enhanced binding in response to reducing conditions (10 mM DTT), whereas in response to oxidative conditions (10 mM H2O2) DNA-binding was reduced (Fig. 4A). This negative effect is reversible as the subsequent addition of 10 mM DTT was able to restore REV DNA binding ... We examined the possibility of whether the C-terminal PAS-domain of REV might act as a redox sensor domain. Redox-DPI-ELISA experiments with HIS-REV lacking the PAS-domain (HIS-REVΔPAS) showed the same redox-sensitive behavior as observed for HIS-REV (Fig. 4B). However, without the PAS-domain, REV-DNA binding was strongly enhanced, supporting the idea that the PAS-domain regulates REV activity via a steric masking mechanism, as proposed by Magnani and Barton (2011). It is conceivable that the observed redox effects in the ELISA system are due to an influence of E. coli proteins on the activity of REV. To exclude such effects, we purified GST-REV protein from E. coli and performed in vitro gel retardation assays in the presence of reducing agents (DTT) and oxidizing agents (H2O2) (Fig. 4C). These gel-shift experiments largely confirm the results obtained by redox-DPI-ELISA and confirm that REV activity can be modulated by the intracellular redox state ... To validate redox-sensitive DNA binding in planta, we treated 35S::FLAG-GR-REVd transgenic plants with either a mock substrate (0.1% ethanol), dexamethasone (DEX) or DEX+0.1% H2O2. In 12-day-old seedlings, we detected REV binding to binding site 2 (fragment II) and no binding was observed to binding site 1 (fragment III). When treated with hydrogen peroxide prior DEX induction, binding to binding site 2 was significantly affected (Fig. 4D), indicating that REV DNA binding is indeed redox sensitive. The same experiment with 7-week-old plants revealed that, at later developmental stages, both binding sites are occupied by REV and the binding seems to be enhanced but exhibits the same redox sensitivity (Fig. 4E). Taken together, we demonstrate that REV shows a stage-specific redox-dependent DNA-binding behavior and that oxidizing conditions decrease the ability to bind DNA in vitro and in vivo

Feature

Additionally, PGM2 and PGM3 proteins from A. thaliana have previously been cloned and expressed in Escherichia coli and the recombinant proteins were analyzed for substrate specificity and affinity. However, no differences between PGM2 and PGM3 were observed

Feature

Additionally, PGM2 and PGM3 proteins from A. thaliana have previously been cloned and expressed in Escherichia coli and the recombinant proteins were analyzed for substrate specificity and affinity. However, no differences between PGM2 and PGM3 were observed

Feature

The Arabidopsis thaliana genome also has another gene, At2g41225, which encodes a protein of only 67 amino acids that phylogenetically belongs to the OSR member (Additional file 1: Figure S1A) [31]. Careful alignment of its amino acid sequence with those of OSR1, ARGOS, and ARL showed that At2g41225 had the conserved LPPLPPPP motif and the C terminal transmembrane helix of the OSR domain. However, it lacked the N terminal transmembrane helix but instead had a plasma membrane-localized signal peptide predicted by Phobius and iPSORT (Additional file 1: Figure S1B) (http://www.ebi.ac.uk//Tools/pfa/phobius webcite; http://ipsort.hgc.jp webcite). Moreover, At2g41225 is located alongside OSR1 in the genome, suggesting that these two genes may originate from a gene duplication event [31]. Therefore, we designated At2g41225 as Organ Size-Related 2 (OSR2).

Feature

DRL1 was reported previously to encode a homolog of the yeast Elongator-associated protein, KTI12 (Cho et al., 2007; Nelissen et al., 2003). To examine the evolutionary history and structural features of the DRL1 protein, we aligned the Arabidopsis DRL1 sequence with the sequences of DRL1 homologs from various other species. Alignment of the amino acid sequences of DRL1 homologs from plants (Arabidopsis thaliana, Oryza sativa, Zea mays, and Glycine max), yeast (S. cerevisiae and Schizosaccharomyces pombe), protozoa (Dictyostelium discoideum), and animals (Danio rerio and Homo sapiens) revealed the presence of conserved domains including an ATP/GTP-binding motif in the N-terminus, two calmodulin (CaM)-binding motifs in the N- and C-terminal regions, and domains specific to plant species (plant-specific sequences I and II) (Fig. 1A). The Arabidopsis DRL1 amino acid sequence exhibited the highest similarity to the sequences of other plant DRL1 homologs with similarities of 58.55, 72.52, and 66.11% to the DRL1 homologs of O. sativa, G. max, and Z. mays, respectively, and the lowest similarity to the yeast DRL1 homologs from S. cerevisiae (28.15%; Fig. 1D). Based on an amino acid alignment and functional site prediction analyses, we identified a conserved sequence, KTQ(R)DVR(K) designated plant-specific sequence I, in the central region that could form a short α-helix and may have WD40 repeat-binding motif (Fig. 1B; Dinkel et al., 2013). We also identified a second invariant sequence, GQS(Y/T)SL designated plant-specific sequence II, in the C-terminal region that was conserved among dicot plants; could form a shorter α-helix than in the yeast proteins and may have NEK2 phosphorylation motif (Fig. 1C; Dinkel et al., 2013). Among the genes examined in this study, the H. sapiens DRL1 homolog encoded the longest amino acid sequence (Fig. 1A). A protein secondary structure prediction analysis of the full amino acid sequences of Arabidopsis (DRL1) and S. cerevisiae (KTI12) using the PHYRE2 program (www.sbg.bio.ic.ac.uk/phyre2/) showed that the structure of both proteins was very similar (Supplementary Fig. S1). The DRL1 protein contained 13 α-helices and 6 β-sheets, while the KTI12 protein had 11 α-helices and 5 β-sheets (Supplementary Figs. S1A–S1D). Based on hydropathy and transmembrane prediction analysis using TMpred and SPLIT programs, the DRL1 and KTI12 proteins apparently lack any transmembrane domains (Supplementary Figs. S1E and S1F

Feature

Furthermore, the kti12Δ mutant is known to exhibit zymocin toxin resistance (Fichtner et al., 2002; Frohloff et al., 2001). KTI12 overexpression also causes zymocin resistance, although the degree of zymocin resistance casued by elevated KTI12 gene expression is lower than that induced by deletion of KTI12 gene (Frohloff et al., 2001). We also performed an assay for zymocin resistance in WT and kti12Δ mutant with or without DRL1 overexpression. Multicopy DRL1 in combination with the GAL1-driven expression of the γ-toxin tRNase subunit from zymocin from vector pHMS14 in galactose medium failed to elicit zymocin resistance, while in multicopy, the yeast KTI12 gene suppressed zymocin and triggered resistance to the tRNase toxin (Fig. 4). This indicates that the function of Arabidopsis DRL1 may not overlap with yeast KTI12 in zymocin mediated growth inhibition

Feature

In addition, the kti12Δ mutant exhibited sensitivity to drugs including caffeine (Fichtner et al., 2002; Frohloff et al., 2001). We performed a complementation assay for caffeine sensitivity in YKL110C, with or without DRL1 expression. The YKL110C mutant was sensitive to caffeine (Figs. 3A–3C) and DRL1 expression did not restore growth performance at 30°C or 39°C (Fig. 3A). To further confirm the caffeine sensitivity, we performed a time-course analysis of cell growth on media containing caffeine with different concentration. Expression of DRL1 restored the growth retardation for 9 h at 0 mM caffeine (Fig. 3C). However, expression of DRL1 did not restore the caffeine sensitivity when grown on 6 mM or 7.5 mM caffeine (Figs. 3B and 3C), indicating that the function of Arabidopsis DRL1 may not entirely overlap with yeast KTI12 in caffeine sensitivity

Feature

A protein BLAST search revealed that SLO3 homologs were found in angiosperms, but not in gymnosperms or nonvascular plants (data not shown). Alignment of SLO3 homologs from Arabidopsis, Populus trichocarpa, Solanum lycopersicum, Glycine max, rice (Oryza sativa), Sorghum bicolor, and maize (Zea mays) revealed that these proteins were highly conserved between dicotyledon and monocotyledon plants

Feature

We used bioinformatics predictions (https://www.cs.colostate.edu/∼approve/) based on a combinatorial amino acid code for RNA recognition by PPR proteins (Barkan et al., 2012) to predict the SLO3 PPR binding sites in nad7 intron 2. We have identified four candidate RNA sequences that SLO3 PPR may bind in nad7 intron 2 (Fig. 7A). Interestingly, three of the four candidate sites are located in the vicinity of intron-exon junctions

Feature

We used bioinformatics predictions (https://www.cs.colostate.edu/∼approve/) based on a combinatorial amino acid code for RNA recognition by PPR proteins (Barkan et al., 2012) to predict the SLO3 PPR binding sites in nad7 intron 2. We have identified four candidate RNA sequences that SLO3 PPR may bind in nad7 intron 2 (Fig. 7A). Interestingly, three of the four candidate sites are located in the vicinity of intron-exon junctions

Feature

The SLO3 PPR contains an amino-terminal sequence to target the protein to mitochondria according to SUBAcon (http://suba3.plantenergy.uwa.edu.au/suba-app/flatfile.html?id=AT3G61360.1), which integrates multiple software predictions

Feature

A serine-threonine-rich region is found near the N terminus of ANT (amino acids 13 to 53) (Figures 8A and 8B). Sequences rich in serine and threonine have been implicated in transcriptional activation (Seipel et al., 1992; Gashler et al., 1993)

Feature

All members identified containing two AP2 domains also have a linker region between the domains that is conserved in both sequence and length (Figure 9B). The ANT linker shows between 52 and 60% amino acid identity with the linker regions of the other multiple AP2 domain proteins with a number of invariant amino acids. The invariant glycine residue indicated in the ANT linker by an asterisk is altered to an aspartic acid in the anf-2 gene product

Feature

The AP2 protein contains two tandemly repeated 68-amino acid motifs designated AP2 domains (Jofuku et al., 1994). These are also found in ANT, and Figure 6 shows that each domain has >50°/o amino acid sequence identity with those in the AP2 protein

Feature

The deduced amino acid sequence of this protein exhibited homology to the conserved amino-terminal membrane-anchoring, pro- line-rich, oxygen- and heme-binding domains of micro- somal cytochrome P-450 (Fig. 5B; 40–90% homology to conserved domains of P-450, as defined by Nebert and Gonzalez 1987). The ROT3 protein appeared, therefore, to include all of the functionally important domains of a P-450 monooxygenase (Pan et al. 1995

Kim GT, Tsukaya H, Uchimiya H - The ROTUNDIFOLIA3 gene of Arabidopsis thaliana encodes a new member of the cytochrome P-450 family that is required for the regulated polar elongation of leaf cells

Feature

CYP90C1 does not have the consensus sequence PFGG(ASV)GRRC(PAV)G around the heme-binding cysteine (Fig. 5B; positions 448–452) that is conserved in members of class A P-450s that cata- lyze plant-specific reactions, an indication that CYP90C1 (non-class A) is more similar to animal, lower eukaryotic, and bacterial proteins (Fig. 5B; Durst and Nelson 1995)

Kim GT, Tsukaya H, Uchimiya H - The ROTUNDIFOLIA3 gene of Arabidopsis thaliana encodes a new member of the cytochrome P-450 family that is required for the regulated polar elongation of leaf cells